Regeneration of biological tissues

Abstract

Disclosed herein are encapsulated microspheres comprising a plurality of cells, e.g., chondrocytes, where the plurality of cells is arranged into clusters. The disclosed encapsulated microspheres recapitulate many features of native articular cartilage and are able to be cultured on cartilage or bone to cure defects in the cartilage. Also disclosed herein are tissue constructs, orthopedic grafts, methods of generating the disclosed encapsulated microspheres, and methods of testing a candidate therapeutic.

Description

MGH 2024-400-02 Quarles ref.125141.04818 REGENERATION OF BIOLOGICAL TISSUES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 667,068, filed July 2, 2024, which is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] N / A SEQUENCE LISTING

[0003] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “125141_04818.xml” which is 13,138 bytes in size and was created on June 24, 2025. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety. BACKGROUND

[0004] Live-cell 3D bioprinting has recently progressed considerably. This holds promise in solving organ shortages and in providing better models to predict drug treatment outcomes prior to clinical trials. However, few studies have proved that bioprinting itself has helped recapitulate native tissue functions. Unlike a 3D-printed construct using only polymers, a bioprinted construct using live cells must go through a post bioprinting process to mature. To positively influence a bioprinted construct to mature toward its native analog tissue, bioprinting itself needs to provide a cue to initiate and maintain tissue genesis during the post-bioprinting culture. However, existing bioprinting techniques largely fail to accomplish this goal.

[0005] Numerous reported successes have focused on generating anatomical tissue shapes at macro-scales. However, tissue functions are determined by cells, which are largely unable to sense the influence of a macro-scale shape. In contrast, as proved by other bioengineering techniques, aMGH 2024-400-02 Quarles ref.125141.04818 micro-scale cellular pattern is an important biophysical signal in the extracellular microenvironment. Cellular patterns determine cell fates and phenotypes and ultimately determine tissue genesis and functions.

[0006] Unfortunately, existing bioprinting techniques are not capable of patterning cells at a micro-scale to provide this favorable cue. Even with the highest cellular resolution, single-cell droplet bioprinting is not suitable for this patterning purpose because it cannot print any hydrogel. Accordingly, there is a need in the art for improved bioprinting techniques, especially, for difficult to print tissues such as articular cartilage. SUMMARY

[0007] In an aspect of the current disclosure, encapsulated microspheres are provided. In some embodiments, the encapsulated microspheres comprise a plurality of cells in a matrix, wherein the plurality of cells is arranged in clusters of cells within the encapsulated microsphere.

[0008] In an aspect of the current disclosure, tissue constructs are provided. In some embodiments, the tissue constructs comprise a plurality of encapsulated microspheres comprising a plurality of cells in a matrix, wherein the plurality of cells is arranged in clusters of cells within the encapsulated microsphere.

[0009] In an aspect of the current disclosure, orthopedic grafts are provided. In some embodiments, the orthopedic grafts comprise a plurality of encapsulated microspheres comprising a plurality of cells in a matrix, wherein the plurality of cells is arranged in clusters of cells within the encapsulated microsphere, affixed to cartilage, a bone, or a bone fragment.

[0010] In an aspect of the current disclosure, methods of generating encapsulated microspheres are provided. In some embodiments, the methods comprise, at an apparatus comprising a first dispensing unit comprising a first nozzle and a second dispensing unit comprising a second nozzle, (a) forming a first droplet at the first nozzle comprising at least one cell and a first matrix and forming a second droplet at the second nozzle comprising a second matrix; (b) dispensing the first droplet onto a substrate and dispensing the second droplet onto the substrate such that the first droplet and the second droplet collide to form an encapsulated microsphere, wherein the second droplet is dispensed at a velocity sufficient to disrupt the arrangement of cells in the first droplet, thereby generating clusters of cells within the encapsulated microsphere, and wherein the firstMGH 2024-400-02 Quarles ref.125141.04818 matrix and the second matrix comprise reagents that form a gel or a solid following the collision of the first droplet and the second droplet.

[0011] In an aspect of the current disclosure, methods of testing a candidate therapeutic are provided. In some embodiments, the methods comprise contacting the candidate therapeutic to at least one encapsulated microspheres comprising a plurality of cells in a matrix, wherein the plurality of cells is arranged in clusters of cells within the encapsulated microsphere. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] FIGS.1A-1C show an illustration of limitations of existing DOD bioprinting technologies. When droplets of bioink are deposited onto a substrate discretely, the diameter of each droplet can be controlled to be less than 100 µm (A1). However, being so small, a droplet may evaporate quickly, and this dramatically impairs cell viability making this approach impractical (A2). Therefore, multiple to a large number of droplets are required to be deposited adjacent to each other to counteract evaporation. However, once a droplet contacts with its neighbor droplets (B1), they merge together in the first layer and in any layers above (B2). Eventually, a construct with a global homogeneous cell distribution forms (B3) thus it is not possible to pattern cells into clusters inside a bioprinted construct at a micro-scale. Another approach is to quickly polymerize a droplet upon deposition to the substrate (C1) or an existing layer (C2) to form one cell cluster per droplet. However, hydrogel interfaces (shown as green lines) are generated between any neighboring droplets. This limits the interaction of cells between different droplets and limits tissue remodeling as well as impairs tissue maturation. In summary, none of the existing DOD bioprinting technologies can be used to practically and usefully pattern cells into clusters as a biophysical signal. Pink: hydrogel, blue dots: cells.

[0014] FIGS.2A-2F show Cell clusters patterning via bioprinting and pattern analysis. (FIG.2A) Illustration of the DVDOD bioprinting process. i) Global and ii) zoom-in view of cellular cluster generation. Blue syringe: chondrocytes, collagen, and fibrin hydrogels. Green syringe: thrombin medium. (FIG. 2B-i): An array of micro-constructs was generated using different bioprinterMGH 2024-400-02 Quarles ref.125141.04818 parameters and images. (FIG.2Bii): Images of individual clusters were processed, and equivalent circular diameters (ECDs) were calculated. (FIG. 2Biii): 20 unique distribution patterns were selected from the generated distribution patterns. (FIG.2Biv): Pattern scores were calculated using different ECD ranges and ranked. (FIG.2Bv): PA-MCTs were bioprinted using parameters of the 20 unique patterns and cultured for 3 weeks. Cartilage-related gene expression was analyzed using qPCR, and the PCR scores were calculated and ranked. (FIG.2Bvi): The best scoring method was selected according to the correlation between PCR and pattern scores. (FIG. 2C) The Empirical Cumulative Distribution Function (ECDF) plots of the selected 20 unique patterns. (FIG. 2D) Statistical analysis shows that none of the two selected patterns are from the same distributions. (FIG.2E) Representative images of patterned cellular clusters within a bioprinted micro-construct. i) Green staining indicates viable cells. ii) Color-coded cellular clusters according to ranges of ECDs. iii) Distance between groups (inscribed circles of areas between cellular clusters). (FIG. 2F) Heatmap of the normalized gene expression (real-time PCR) and the correlated PCR scores of COL-II, Sox-9, ACAN, and COL-I of the PA-MCTs of the 20 unique patterns.

[0015] FIGS.3A-3C show area percentage bar plots, raincloud plots and ECDF plots of 20 unique patterns (three replicates of each pattern). ECD ranges between 20 and 80 µm, the selected optimized range, are shown in red.

[0016] FIGS. 4A-4B show selection of the scoring method for the best prediction of chondrogenesis. (FIG.4A-i) Overview of the hybrid graph characterizing cell clusters. It features a bar plot for area percentage and a violin plot coupled with a dot plot—collectively known as a raincloud plot—for illustrating the equivalent circular diameter (ECD) distribution and number percentage. (FIG. 4A-ii) Bar plots of the area percentage and raincloud plots of the number percentage of 20 unique patterns. (FIG.4B-i) Heatmap of the rankings of PCR scores and pattern scores of 20 unique patterns using 66 different ECD ranges. (FIG. 4B-ii) Bar plots of the Spearman's correlations between PCR score ranking and pattern score rankings. Dash lines demonstrate the pattern ranking using the ECD range of 20–80 µm has the highest correlation with PCR ranking. (PAT: Pattern).

[0017] FIG.5 shows a schematic overview of MCT development processes and applications after the patterning method is developed: Bioprinting, Bio-assembling, Characterization, and Application. This scheme encapsulates the core methodologies and applications reported in theMGH 2024-400-02 Quarles ref.125141.04818 article. (1) Characterizing individual PA-MCTs (vs. H-MCTs) and their cartilagenesis, (2) characterizing the bio-assembly process of PA-MCTs (vs. H-MCTs) and the accompanying cartilagenesis, (3) characterizing PA-MCTs (vs. H-MCTs) integrating with native articular cartilage and bone tissues and the accompanying cartilagenesis, (4) characterizing cartilage defect repair with PA-MCTs (vs.40-28 H-MCTs) and accompanying cartilagenesis in an osteochondral explant model, (5) generating personalized allografts, (6) resurfacing an entire femoral condyle, (7) establishing OA-PA-MCTs and screening candidate drugs for DMOADs, (8) using PA-MCTs as a miniature healthy articular cartilage model. PA-MCTs: patterned articular micro-cartilage- tissues; H-MCTs: (cells) homogeneously distributed micro-cartilage-tissues; DMOADs: disease- modifying osteoarthritis drugs.

[0018] FIGS.6A-6H show (FIG.6A) Schematic of patterned clusters serving as a biophysical cue. (FIG. 6B-i,ii) Typical images of PA-MCTs: (FIG.6B-i) fluorescence (vs H-MCTs), (FIG. 6B-ii) phase contrast. (FIG. 6B-iii) Chondrocytes in PA-MCTs remain highly viable over 5 weeks (CellTiter-Glo assays, *p < 0.05 versus H-MCTs by independent-samples t-test). (FIG. 6C) Cartilagenesis in individual PA-MCTs. Typical native-comparable morphology of articular cartilage i): round chondrocytes, isogenous groups, and lacunae (Safranin-O staining). sGAG (Safranin-O staining) and collagen type II (ii, immunofluorescence staining) were highly expressed in PA-MCTs. (FIG.6D) Higher levels of chondrogenic gene expression and lower levels of fibrotic gene expression were detected in PA-MCTs (*p < 0.05 vs H-MCTs by independent- samples t-test) (FIG. 6E-i) The developed PA-MCTs are spherical and demonstrate typical cartilage white appearance (5 weeks, stereo-microscopy, hash mark = 1 mm). (FIG. 6E-ii) PA- MCTs are injectable through a glass capillary tubing (≈1.4 mm ID) and a plastic injector (≈2.5 mm ID). (FIGS.6F, 6G) Quantification of sGAG (FIG.6F) and collagen type II (FIG.6G) (*p < 0.05 vs H-MCTs and native articular cartilage, by ANOVA). (FIG. 6H) The stiffness of PA-MCTs is significantly higher than H-MCTs (By micropipette-aspiration; *p < 0.05 by independent-samples t-test). All data are means ± SD, n ≥ 3 in all groups. NC: native cartilage; PA-MCT: patterned articular micro-cartilage tissue; H-MCT: homogeneously distributed micro-cartilage tissue.

[0019] FIG.7 shows high-resolution representative images of typical Safranin O staining of PA- MCTs and H-MCTs. Scale bars = 100 µm. Same images at lower resolution are shown in FIG.6C.MGH 2024-400-02 Quarles ref.125141.04818

[0020] FIG. 8 shows PA-MCTs demonstrate significantly higher Modified Bern Score than H- MCTs. n ≥ 3, data are means ± SD, *** P < 0.001 vs. H-MCTs by independent-samples t test.

[0021] FIGS.9A-9G show characterizing the cartilagenesis during the bio-assembly of PA-MCTs versus H-MCTs. (FIG. 9A) Schematic of the process: individual PA-MCTs were cultured for 2 weeks, and multiple PA-MCTs were placed together to assemble for a total of 7 weeks. (FIG.9B) A representative image demonstrating chondrocytes migrating from a PA-MCT into the surrounding hydrogel after 24 h. (FIG.9C) Representative histological images demonstrating the progress of cartilagenesis during the bio-assembly of PA-MCTs, and darker blue indicates a higher level of sGAG expression. The width of the color bar approximates the ratio of the indicated activity. (FIG. 9D) Representative images of the gross appearance of the assembled macro- articular-cartilage over time. (FIG. 9E) A representative image of a completed macro-articular- cartilage demonstrating robust sGAG expression. (FIG. 9F) Representative histological and immunohistochemical images of large macro-articular-cartilage assembled from PA-MCTs i): High magnification images demonstrate typical round chondrocytes, isogenous groups, and lacunae; darker red indicates a higher level of sGAG expression. The assembly from H-MCTs was not intact and visible holes exist ii). (FIG. 9G) PA-MCTs demonstrated significantly higher Young's modulus and aggregate modulus, and a significantly lower permeability constant than H- MCTs, indicating more mature—more solid and less porous—internal structures in PA-MCTs. (n ≥ 3, data are means ± SD, *p < 0.05 by ANOVA.).

[0022] FIGS.10A-10B show Demonstration of the early stage of PA-MCTs assembly (FIG.10A) a phase-contrast image; (FIG.10B): a section stained with Alcian blue). PA-MCTs were cultured for 2 weeks and then collected and assembled. The images represent the status of week 4 of the total culture period. The blue color intensity of Alcain blue staining is linear to GAG content. Chondrocytes migrated from the insides of individual PA-MCTs into spaces between them where, as indicated by lighter blue, the GAG deposited by the migrated chondrocytes was relatively low at the early stage. Scale bars = 100 µm.

[0023] FIGS. 11A-11C show semi-quantification of GAG content inside and outside PA-MCTs during cartilagenesis of bio-assembly. (FIG.11A) A representative image of Alcian blue staining of a PA-MCT and the matrix around it at week 3 of the total culture period. Red and blue dashed regions indicate areas inside and outside the PA-MCT. Scale bar = 50 µm. The image is also shownMGH 2024-400-02 Quarles ref.125141.04818 in FIG 4C. (FIG.11B) GAG content curves. Pixel intensities, representing relative GAG content, along a series of parallel lines across the image were analyzed. The height of each curve represents the level of GAG content. Y: Pixel intensity value, bar = 100; X: Distance, bar = 100 pixels. (FIG. 11C) Average GAG content inside PA-MCTs is significantly higher than outside PA-MCTs. n ≥ 3, data are means ± SD, * P < 0.05 by independent-samples t test.

[0024] FIG. 12 shows a representative histological image in high-resolution of an assembled macroarticular-cartilage tissue (week 7 of the total culture period, a panoramic view). PA-MCTs demonstrate the capability of cartilagenesis during the assembly process. Seamless histological assembly and extensive GAG expression throughout were visualized. Scale bar = 1 mm. The same image at a lower resolution is shown in FIG.9F.

[0025] FIG. 13 shows cartilagenesis during integration with native articular cartilage and bone using PA-MCTs versus H-MCTs. All PA-MCTs and H-MCTs were cultured individually for 2 weeks before integration assays. PA-MCTs demonstrate the capability of cartilagenesis. Macroscopy: (FIG. 13-i) When integrating into native cartilage and bone, PA-MCTs were individually visible at week 5 and assembled to intact macro-articular-cartilage with smooth and white surfaces at week 7. (FIG. 13-ii) H-MCTs only partially assembled and adhered to native cartilage, and adhered loosely to or fell off native bone (arrow). Microscopy: (FIG. 13-iii) PA- MCTs seamlessly and histologically integrated with native cartilage and bone with intense sGAG expression indicated by dark blue staining. (FIG.13-iv) Cartilagenesis did not occur in H-MCTs assembly, and weak sGAG was expressed as indicated by light blue staining.

[0026] FIG. 14 shows sGAG expression in the integration assay at week 7 of the total culture period. PAMCT-assemblies demonstrated significantly higher GAG content than H-MCTs- assemblies evaluated by semi-GAG-quantification. n ≥ 3, data are means ± SD, * P < 0.05, ** P < 0.01 vs. H-MCTs by independent-samples t test.

[0027] FIGS. 15A-15J show cartilagenesis during cartilage defect repairing using PA-MCTs versus H-MCTs. All PA-MCTs and H-MCTs were cultured individually for 2 weeks before defect- repairing assays. (FIG. 15A) Schematic of the defect repair process. (FIG. 15B, FIG. 15C) PA- MCTs demonstrate the capability of cartilagenesis when repairing cartilage defects in an osteochondral model. Both full-thickness and partial-thickness defects were completely repaired by the PA-MCT-assembly forming neocartilage with a smooth white surface (FIG. 15B).MGH 2024-400-02 Quarles ref.125141.04818 Microscopically the PA-MCT-assembly demonstrates seamless structural integration at all locations (FIG. 15C): an irregular cartilage wall interface i), the bone interface ii), the partial- thickness defect interface iii), the deep region in the neocartilage iv). (FIG. 15D, FIG. 15E) H- MCTs failed to repair the defect. A large proportion of the defect was void due to unassembled H- MCTs fell off the explant during culture and a large degree of H-MCT compaction (FIG.15D-i) and sliced views / (FIG. 15D-ii). Necrosis, compaction of H-MCTs, and void spaces underneath the neo-tissue surface were observed histologically (FIG.15E). (FIG.15F) Semi-quantification of sGAG content: yellow, green, and cyan dotted regions correspond to native cartilage, the result of full-thickness defect repair and partial-thickness defect repair, respectively. The intensity of every pixel in the red channel, ranging from 0 to 255 (color bar), represents the level of sGAG expression. The intensity-distribution map demonstrates intense sGAG expression throughout the neocartilage, including the deep zone. (FIG.15G) Preliminary zonal structure formed in the PA- MCT-assembly, which is similar to that in the native articular cartilage. (FIG. 15H) PA-MCT- assemblies demonstrated significantly higher adhesion strength to the host cartilage than H-MCT- assemblies in the push-out test. n ≥ 3, data are means ± SD, *p < 0.05 by ANOVA. (FIG.15I, FIG. 15J) Proof-of-concept repairing a large irregularly-shaped cartilage defect in a sheep femoral condyle (FIG. 15I-i) and resurfacing an entire bare bone rabbit femoral condyle (FIG. 15J-i) by the PA-MCT-bio-assembly. All PA-MCTs were cultured for cartilagenesis individually and implanted at week 2. Both demonstrate similar progress: individual PA-MCTs were clearly visible at implantation (FIG. 15I-ii, ≈800 PA-MCTs); boundaries between PA-MCTs gradually became blurry (FIG.15J-iii), and smooth white cartilage surfaces were regenerated at week 7 that defects were repaired (FIG.15I-iii) and the femoral condyle bone was resurfaced (FIG.15J-iv).

[0028] FIGS.16A-16D show a demonstration of the delivery of PA-MCTs through injection. PA- MCTs with an average diameter of ~≤ 650 µm can be manually injected through a rigid tube as small as 1.4 mm ID smoothly by applying low pressure on the plunger. FIG. 16A shows a microscopic image of PA- 40-38 MCTs inside the glass tubing with a 1.4 mm ID. FIG.16B shows the injection device (~2.5 mm ID and ~3.3 mm OD) used for delivering PA-MCTs to the cartilage defects. The device's OD is smaller than the OD (4 mm) of a typical arthroscope. Therefore, the device can be suitable to be inserted into the human knee to inject PA-MCTs to the defect in a potential minimally invasive surgery. FIG. 16C and FIG. 16D show the completion of injectingMGH 2024-400-02 Quarles ref.125141.04818 ~230 PA-MCTs to a defect created in a femoral condyle of a sheep knee. Scale bars = 500 µm (FIG.16A), 1 cm (FIG.16C), 3mm (FIG.16D).

[0029] FIG. 17 shows PA-MCTs demonstrated significantly higher Modified O'Driscoll Scores than HMCTs in repairing osteochondral defects (week 7 of the total culture period). n ≥ 3, data are means ± SD, *** P < 0.001 vs. H-MCTs by independent-samples t test.

[0030] FIGS.18A-18B show a demonstration of the capability of PA-MCTs for self-fitting curved surfaces. To evaluate the capability of PA-MCTs self-fitting curved surfaces of an articular joint for potential clinical implantation, silicone sheets with curved edges were used to mimic joint curves. Silicone sheets with convex (FIG.18A) or concave (FIG.18B) edges were placed between two glass slides. PA-MCTs were gently placed over each surface, and PA-MCTs self-fitted each curvature and attached to each surface seamlessly. Scale bars = 1 mm.

[0031] FIGS. 19A-19M show generating personalized osteochondral allografts. Implantation of an osteochondral rather than cartilage-only allograft is necessary for cartilage defect diseases accompanied by an 40-40 underlying bone lesion, such as osteochondritis dissecans. Natural allografts have the drawbacks of not fitting the geometry of a host knee and are of limited availability. To meet the clinical demand and provide a potential solution for osteochondritis dissecans lesions, here we present a proof-of-concept methodology generating personalized allografts using PA-MCTs and decellularized bone tissues. Engineered allografts were designed to fit the patient's dimensional information of the mimicked diseased knee joint in the following 4 steps. (i) Generating a healthy osteochondral model of a distal femur (FIGS. 19A–19E): regions of the femur bone were segmented from computed tomography images of a human joint (FIGS. 19A–19C); the distal femur bone was volumetrically 3D reconstructed (FIG.19D); and a cartilage layer was created from the bone surface (FIG> 19E). (ii) Cartilage defect creation and allograft design (FIGS.19F–19I): a defect was created on the weight-bearing region of the medial condyle (FIG.19F, shown in red), and chondral lesion preparation was mimicked in the model (FIG.19G). An osteochondral allograft was designed to fit the defect (FIG.19H, dashed line region; FIG.19J, magnified view) and implantation was also mimicked (FIG.19I). (iii) Generating an osteochondral allograft: according to the dimensional Information of the model (FIG. 19J), a cylindrical bone with a curved top was created and decellularized. Approximately 700 PA-MCTs were placed on top of the bone, where individual PAMCTs were visible at week 1 (FIG.19K). At week 7 of theMGH 2024-400-02 Quarles ref.125141.04818 total culture period, an osteochondral allograft (FIG. 19L) was generated with high shape and structural fidelity, matching the dimensional Information of the patient knee (FIG. 19J). The generated allograft has a smooth and white articular surface. Safranin O staining shows intense GAG expression and typical articular cartilage morphology in the cartilage layer of the allograft (FIG.19M). Scale bars = 2 mm (FIG.19K, FIG.19L), 500 µm (FIG.19M).

[0032] FIGS.20A-20H show establishment of OA-PA-MCTs model using IL-1β stimulation and application of PA-MCTs as an articular cartilage development model. (FIG. 20A) PA-MCTs demonstrated a smaller variation than all groups of explants (from the entire knee or a region; MTP: medial tibial plateau; LTP: lateral tibial plateau; MFC: medial femoral condyle; LFC: lateral femoral condyle). (FIG. 20B) An OA-PA-MCTs model was created by IL-1β stimulation, and significant sGAG loss was observed. (FIG.20C) The feasibility of the model was further evaluated by analyzing the disease-modifying effects of dexamethasone, Celecoxib, IGF-1, and TGF-β on the OA-PA-MCTs, including the analyses of sGAG content and gene expression. (FIG.20D) PA- MCTs were used as a miniature model of healthy cartilage. FITC-dextran was used for mimicking a gene carrier and was successfully microinjected into PA-MCTs. (FIG.20E) Characterizing PA- MCTs and H-MCTs bioprinted with hMSCs. hMSCs (green) were patterned into clusters separated by the hydrogel matrix (red, reflection confocal) 3 days postbioprinting. After 5 weeks of chondrogenic differentiation, hMSCs showed native-chondrocyte-like morphology and lacuna microstructure in PA-MCTs while elongated fibroblast-like morphology in H-MCTs. (FIG. 20F) sGAG content in PA-MCTs was significantly higher than that in H-MCTs. (FIG. 20G) All the expression of chondrogenic phenotype genes in PA-MCTs were higher than those in H-MCTs, and notably, collagen type II was significantly higher, while fibrotic phenotype gene, collagen type I, was significantly lower (quantitative PCR, all data were normalized to those of 18S rRNA). (FIG. 20H) PA-MCTs bioprinted with hMSCs were also able to bio-assemble into macro-cartilage as demonstrated by dynamic tracking and histology. All data are mean ± SD, except for Panel A, which shows coefficients of variation (CVs) as single values. #: significantly different from control, $: significantly different from IL-1β,*p < 0.05, Δ p < 0.01, Ø p < 0.001, by ANOVA (FIG. 20A, FIG.20C. The bar plots in FIG.20A illustrate the CV, while the statistical analysis—based on sGAG content values among groups—is indicated on the plot in a hybrid manner.), by independent-samples t-test FIG.20F, FIG.20G). OA: osteoarthritis.MGH 2024-400-02 Quarles ref.125141.04818

[0033] FIG.21 shows an illustration of the process of cartilage defect repair by the bio-assembly of PA-MCTs or H-MCTs in an osteochondral explant model. One corner of each explant was removed for ease of sample orientation during observation.

[0034] FIG.22 shows an illustration of the micropipette aspiration assay.

[0035] FIGS.23A-23D show photographs of the device (FIG.23A, FIG.23C) and schematics of the mechanical tests (FIG.23B, FIG.23D). Samples were tested using unconfined (FIG.23A, FIG. 23B) and confined (FIG. 23C, FIG. 23D) compression tests in a testing chamber supplemented with a saline solution (FIG.23A, FIG.23C), which was omitted while being photographed.

[0036] FIGS.24A-24D show an illustration of the push-out assay.

[0037] FIG. 25 shows an example process to analyze encapsulated microspheres in accordance with some embodiments of the disclosure.

[0038] FIG. 26 shows an example system configured to analyze encapsulated microspheres in accordance with some embodiments of the disclosure. DETAILED DESCRIPTION

[0039] Disclosed herein are encapsulated microspheres, tissue constructs comprising the encapsulated microspheres, orthopedic grafts comprising the encapsulated microspheres, methods of generating encapsulated microspheres, and methods of testing a candidate therapeutic. Encapsulated microspheres

[0040] The inventor discovered a new bioprinting technique termed directly controlled droplet bioprinting (DVDOD). Using DVDOD, the inventor can pattern cells into multiple clusters within a droplet of bioink. The inventors demonstrated that the patterning of cells into clusters, similar to the native isogenous clusters found in articular cartilage, is sufficient to support cartilagenesis.

[0041] Accordingly, in an aspect of the current disclosure, encapsulated microspheres are provided. In some embodiments, the encapsulated microspheres comprise a plurality of cells in a matrix, wherein the plurality of cells are arranged in clusters of cells within the encapsulated microsphere.MGH 2024-400-02 Quarles ref.125141.04818

[0042] The cells may comprise any cell, e.g., cells of connective tissue, e.g., cells of cartilage, e.g., cells of articular or non-articular cartilage or cells of hyaline cartilage, elastic cartilage, or fibrocartilage. The cells may include, but are not limited to, chondrocytes, mesenchymal stem cells (MSCs), or a combination thereof.

[0043] The cells may be vertebrate, e.g., mammal, e.g., human, dog, cat, mouse, rat, horse, donkey, sheep, etc., cells.

[0044] Encapsulated microspheres are spherical to spheroid in shape with size on the order of 1 ^m to about 1000 ^m, or any subrange or value therein. Encapsulated microspheres have a matrix that is solid, semi-solid, or a gel with an outside interface that prevents cells from moving outside the matrix by random movement. Thus, cells are “encapsulated” in the microspheres. Further, the matrix may be sufficiently dense or viscous to prevent movement of the cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 days or more after formation of the encapsulated microspheres (e.g., FIGs.9A, 9B, and 9C).

[0045] In the working examples, the inventor demonstrates a two-part approach to generating a matrix that encapsulates the plurality of cells, where fibrin is incorporated into a first droplet and thrombin is incorporated into a second droplet. Physical force from the second droplet contacting the first droplet disperses the plurality of cells into clusters and generates a solid or semi-solid fibrin matrix that locks the clustered cells into place without introducing an interface between the clusters (FIG.2A).

[0046] As used herein, “clusters” of cells refers to at least 2 cells, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more cells in proximity to each other such that the at least 2 cells are not uniformly or homogenously distributed within the matrix, as compared to the other cells in the plurality of cells in the encapsulated microsphere. The inventor demonstrated, using a control system that produces homogenously distributed cells, e.g., chondrocytes (H-MCTs), that homogenous distribution of cells does not recapitulate features of native arthritic cartilage (e.g., FIGs.6C, 6F, 6H).

[0047] Homogenously distributed cells within encapsulated microspheres may be generated by first applying a cell-free thrombin-containing droplet to a substrate and contacting the thrombin- containing droplet with a droplet containing fibrin and a plurality of cells at a low velocity. FIG.MGH 2024-400-02 Quarles ref.125141.04818 6B shows a comparison of PA-MCT (clustered cells) vs. (H-MCT) homogenous cell distribution. Statistical methods of determining / classifying the distribution of cells within a matrix as clustered vs. homogenous are known in the art.

[0048] The encapsulated microspheres comprise a plurality of cells, e.g., 2 to about 1x106cells, e.g., chondrocytes, mesenchymal stem cells (MSCs), or any subrange or value therein. The plurality of cells may comprise about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, or more cells.

[0049] The encapsulated microspheres may be spheroid or substantially spheroid and have a diameter of about 1 ^m to about 1 mm. In some embodiments, the encapsulated microspheres may have a diameter of about 100 ^m to about 700 ^m or greater than about 400 ^m to less than about 650 ^m.

[0050] The inventor evaluated the equivalent circular diameter (ECD) of clusters of cells, which is the diameter of a circle that has the same area as a non-circular particle, i.e., a cluster of cells, and found that clusters with ECD ≤ 20 µm can form spontaneously, while clusters with an ECD of about 220 µm or greater encompassed samples containing large homogenous clusters. Accordingly, the clusters of cells may have an average equivalent circular diameter of about 20 ^m to about 200 ^m in the encapsulated microspheres.

[0051] The matrix may further comprise a cell growth or maintenance medium, e.g., Dulbecco’s modified Eagle medium (DMEM), RPMI, RPMI 1640. The medium may comprise serum, e.g., fetal bovine serum (FBS) or may be serum-free. Serum-free media may comprise, e.g., recombinant albumin, e.g., recombinant human albumin, or another suitable substitute, several of which are known in the art. The medium may be a defined medium, i.e., each of the components of the medium can be individually described by chemical structure. Defined media are known in the art and the selection of the appropriate media is within the skill of an ordinary artisan. Good manufacturing practices (GMP) medium may be used, especially, in the generation of tissue constructs or orthopedic grafts for transplantation into a subject, e.g., a human subject.

[0052] The plurality of cells in the encapsulated microsphere may further comprise an exogenous polynucleotide. As used herein, “exogenous” refers to a polynucleotide originating from outsideMGH 2024-400-02 Quarles ref.125141.04818 the plurality of cells, e.g., an expression construct. The exogenous polynucleotide may comprise a sequence encoding a polypeptide. The polypeptide may comprise, e.g., a nucleic acid-guided nuclease, e.g., a Cas nuclease, e.g., a Cas9 nuclease. The exogenous polynucleotide may comprise a sequence encoding a guide RNA. The exogenous polynucleotide may comprise sequences encoding proteins including, but not limited to, collagen peptides such as type 2 collagen, aggrecan or aggrecan peptides, or other structural proteins (e.g., peptides from fibronectin, cartilage oligomeric matrix protein (COMP)), signaling peptides, e.g., signaling peptides derived from growth factors including, but not limited to, bone morphgenic proteins (BMPs) or transforming growth factor beta 3 (TGF-^3). Tissue constructs

[0053] The inventor demonstrated that the disclosed encapsulated microspheres can be used to generate macro-scale (on the order of millimeters) constructs that resemble articular cartilage (FIG. 9). Accordingly, in an aspect of the current disclosure, tissue constructs are provided. In some embodiments, the tissue constructs comprise a plurality of the disclosed encapsulated microspheres.

[0054] A plurality of encapsulated microspheres may comprise about 3 to about 100, about 1000, about 10000, or more encapsulated microspheres, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 encapsulated microspheres, or any value or subrange therein.

[0055] The tissue construct may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mm or more in diameter, or any subrange or value therein (“macroscale”). The inventor demonstrated the exemplary generation of a 9 mm tissue construct (FIGs.9D and 9E), with smooth white surfaces and sulfated glycosaminoglycan (sGAG) deposition throughout the construct. The deposition of sGAG can be detected by methods known in the art, e.g., alcian blue staining. Orthopedic graftsMGH 2024-400-02 Quarles ref.125141.04818

[0056] The inventor grafted the disclosed encapsulated microspheres directly onto bone or directly onto cartilage with full-thickness or partial-thickness defects (FIGs. 13-15). Accordingly, in an aspect of the current disclosure, orthopedic grafts are provided. In some embodiments, the orthopedic grafts comprise a plurality of the disclosed encapsulated microspheres affixed to cartilage, a bone, or a bone fragment.

[0057] The inventor deposited the encapsulated microspheres onto defects in cartilage or bone and covered them in a solidifying matrix comprising fibrin activated by thrombin. The encapsulated microspheres may be affixed to the cartilage, bone, or bone fragment in a matrix comprising fibrin.

[0058] The cartilage or bone may be decellularized. Decellularized bone may be produced by methods known in the art and those shown in the working examples. Briefly, the tissues, e.g., cartilage or bone, may be exposed to a detergent, e.g., sodium dodecyl sulfate (SDS) and rocked or shaken for a suitable amount of time to remove cells to generate decellularized tissue. The decellularized tissue may be contacted with a sanitizing agent, e.g., 70% ethanol, prior to use.

[0059] Also disclosed are methods of generating the orthopedic grafts, comprising the reagents and steps described above for generating the encapsulated microspheres as well as the method steps and reagents for affixing the encapsulated microspheres to a substrate, e.g., cartilage, a bone, or a bone fragment. Methods of generating encapsulated microspheres

[0060] As discussed above and demonstrated in the drawings and Examples, the inventor discovered methods to produce encapsulated microspheres comprising clusters of cells, which is sufficient to recapitulate the unique features of articular cartilage (e.g., FIGs.2A and 9D, 9E, 9F, and 9G).

[0061] Accordingly, in an aspect of the current disclosure, methods of generating encapsulated microspheres are provided. In some embodiments, the methods comprise, at an apparatus comprising a first dispensing unit comprising a first nozzle and a second dispensing unit comprising a second nozzle, (a) forming a first droplet at the first nozzle comprising at least one cell and a first matrix and forming a second droplet at the second nozzle comprising a second matrix; (b) dispensing the first droplet onto a substrate and dispensing the second droplet onto the substrate such that the first droplet and the second droplet collide to form an encapsulatedMGH 2024-400-02 Quarles ref.125141.04818 microsphere, wherein the second droplet is dispensed at a velocity sufficient to disrupt the arrangement of cells in the first droplet, thereby generating clusters of cells within the encapsulated microsphere, and wherein the first matrix and the second matrix comprise reagents that form a gel or a solid following the collision of the first droplet and the second droplet.

[0062] An exemplary bioprinter for use in the disclosed methods is described in U.S. Pat. No. 11,400,183, which is incorporated by reference herein in its entirety.

[0063] The first and second dispensing unit may each comprise a syringe including (i) a hollow body and a plunger dimensioned to translate in the body, the body having an exit orifice; (ii) an actuator in contact with a proximal end of the plunger; (iii) a controller for controlling linear motion of the actuator wherein the controller executes a program received from a computer in the controller to drive the actuator toward the proximal end of the plunger to dispense a bioink from the body of the syringe; and (iv) a nozzle having a wall defining a fluid path extending from an inlet of the nozzle to an outlet of the nozzle, the inlet of the nozzle being in fluid communication with the exit orifice of the body of the syringe, wherein as the controller drives the actuator toward the proximal end of the plunger bioink flows into the fluid path, wherein the nozzle includes a fluid passageway in fluid communication with a source of fluid and the fluid path, wherein the source of fluid comprises a controllable valve for supplying pulsed fluid from the source of fluid to the fluid passageway to create separate droplets from the flow of bioink in the fluid path in the nozzle by blowing the droplets away from the nozzle using the pulsed fluid from the source of fluid.

[0064] The first matrix may comprise fibrin and the second matrix may comprise thrombin or the first matrix may comprise thrombin and the second matrix may comprise fibrin.

[0065] The first droplet may be dispensed such that the cells achieve a relatively homogenous distribution in the droplet. This may be achieved, in a system that uses pulsed fluid, e.g., pulsed air, to blow the first droplet from the first nozzle onto the substrate, by using a low-pressure fluid pulse. The second droplet may then be blown from the second nozzle using a higher-pressure fluid pulse.

[0066] The pulsed fluid may be a pulsed gas, e.g., pulsed air, e.g., millisecond pulsed air. A volume of each droplet may be in a range of about 10 nanoliters to about 15 microliters, or any subrange or value therein.MGH 2024-400-02 Quarles ref.125141.04818

[0067] The cells may comprise chondrocytes. The cells may comprise MSCs or chondrocytes and MSCs. The first or the second matrix may comprise collagen type I.

[0068] The method may further comprise repeating steps (a) and (b) n additional times, wherein n is an integer greater than 1, to generate n encapsulated microspheres, e.g., n may be 1 to 10000 or any subrange or value therein. n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or more.

[0069] The first droplet may comprise about 2 to about 1x106chondrocytes.

[0070] The parameters of fluid ejection from the first and second nozzle may be varied by the skilled person to achieve a desired volume of droplet.

[0071] The encapsulated microspheres comprise a plurality of cells, e.g., 2 to about 1x106cells, e.g., chondrocytes, mesenchymal stem cells (MSCs), or any subrange or value therein. The plurality of cells may comprise about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, or more cells.

[0072] The encapsulated microspheres may be spheroid or substantially spheroid and have a diameter of about 1 ^m to about 1 mm. In some embodiments, the encapsulated microspheres may have a diameter of about 100 ^m to about 700^m or greater than about 400 ^m to less than about 650 ^m.

[0073] The inventor evaluated the equivalent circular diameter (ECD) of clusters of cells, which is the diameter of a circle that has the same area as a non-circular particle, i.e., a cluster of cells, and found that clusters with ECD ≤ 20 µm can form spontaneously, while clusters with an ECD of about 220 µm or greater encompassed samples containing large homogenous clusters. Accordingly, the clusters of cells may have an average equivalent circular diameter of about 20 ^m to about 200 ^m in the encapsulated microspheres.

[0074] The matrix may further comprise a cell growth or maintenance medium, e.g., Dulbecco’s modified Eagle medium (DMEM), RPMI, RPMI 1640. The medium may comprise serum, e.g., fetal bovine serum (FBS) or may be serum-free. Serum-free media may comprise, e.g.,MGH 2024-400-02 Quarles ref.125141.04818 recombinant albumin, e.g., recombinant human albumin, or another suitable substitute, several of which are known in the art.

[0075] The plurality of cells in the encapsulated microsphere may further comprise an exogenous polynucleotide. As used herein, “exogenous” refers to a polynucleotide originating from outside the plurality of cells, e.g., an expression construct. The exogenous polynucleotide may comprise a sequence encoding a polypeptide. The polypeptide may comprise, e.g., a nucleic acid-guided nuclease, e.g., a Cas nuclease, e.g., a Cas9 nuclease. The exogenous polynucleotide may comprise a sequence encoding a guide RNA.

[0076] The substrate may be a bone or a defect in cartilage, e.g., a partial-thickness defect or a full-thickness defect. A full-thickness defect is defined as a defect in cartilage that exposes the underlying bone.

[0077] The inventor showed that more than one of the disclosed encapsulated microspheres (PA- MCTs), e.g., a plurality, as defined herein, can be placed in proximity to allow chondrocyte migration, new matrix deposition, and reorganization, leading to cartilagenesis (e.g., FIG.9A, 9B, and 9C. At least 2 microspheres may be arranged on a substrate to generate a tissue construct precursor.

[0078] The encapsulated microspheres may be affixed to cartilage, bone, or a bone fragment. The encapsulated microspheres may be delivered to the cartilage, bone, or bone fragment in a delivery matrix comprising fibrin; a solidifying matrix, comprising thrombin, may then be applied to the delivery matrix to solidify the fibrin.

[0079] The methods may further comprise repeating steps (a) and (b) n times and varying at least one parameter of encapsulated microsphere formation in each of the n repeats, to generate n encapsulated microspheres with varying properties; analyzing at least one parameter related to cartilagenesis in each of the n encapsulated microspheres; and selecting at least one parameter of encapsulated microsphere formation, based on the analysis of the at least one parameter related to cartilagenesis.

[0080] The at least one parameter of encapsulated microsphere formation may comprise pulsed fluid pressure of the first and or second nozzle. The at least one parameter related to cartilagenesis may comprise equivalent circular diameter (ECD) (FIG. 2B) of the clusters in the encapsulatedMGH 2024-400-02 Quarles ref.125141.04818 droplet or expression of at least one gene or protein (FIG.2B). Expression of at least one gene or protein may be determined using methods known in the art, e.g., quantitative polymerase chain reaction (qPCR), mass spectroscopy, liquid chromatography coupled to mass spectroscopy (LC- MS), enzyme-linked immunosorbent assay (ELISA), etc.

[0081] The at least one gene or protein may comprise collagen type II (COL-II), transcription factor SOX-9 (SOX9), aggrecan (ACAN), or collagen type I (COL-I), either individually, or in any combination. COL-II, SOX9, and ACAN may be positively related to cartilagenesis and COL- I may be negatively related to cartilagenesis.

[0082] An exemplary flow chart of the disclosed process is illustrated in FIG.25.

[0083] The analysis of the n encapsulated microspheres may be performed at a computer system. An exemplary computer system is shown in FIG.26.

[0084] Systems for producing and analyzing the encapsulated microspheres are disclosed herein. The systems may comprise the bioprinting apparatuses coupled to a computer system. Methods of testing a candidate therapeutic

[0085] The inventors demonstrated that the disclosed encapsulated microspheres (PA-MCTs) can be used as a surrogate for cartilage when testing disease modifying therapies, e.g., disease modifying therapies for osteoarthritis (disease modifying osteoarthritis drugs (DMOADs)) (FIG. 20). Accordingly, in an aspect of the current disclosure, methods of testing a candidate therapeutic are provided. In some embodiments, the methods comprise contacting the candidate therapeutic to at least one of the disclosed encapsulated microspheres.

[0086] The methods may further comprise detecting at least one parameter associated with performance of the candidate therapeutic. The at least one parameter may comprise detecting gene expression, protein expression, glycosaminoglycan (GAG) deposition, or protein secretion. The method may further comprise contacting the at least one encapsulated microsphere with an inflammatory reagent, e.g., interleukin 1 beta (IL-1^). Additional definitions

[0087] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”MGH 2024-400-02 Quarles ref.125141.04818

[0088] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0089] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0090] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0091] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0092] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of theMGH 2024-400-02 Quarles ref.125141.04818 above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Illustrative embodiments 1. An encapsulated microsphere comprising a plurality of cells in a matrix, wherein the plurality of cells are arranged in clusters of cells within the encapsulated microsphere. 2. The encapsulated microsphere of embodiment 1, wherein the clusters of cells are dispersed within the encapsulated microsphere without an interface separating the clusters of cells. 3. The encapsulated microsphere of any one of embodiments 1 or 2, wherein the matrix comprises a fibrin matrix. 4. The encapsulated microsphere of any one of the preceding embodiments, wherein the plurality of cells comprises chondrocytes, mesenchymal stem cells, or a combination of chondrocytes and mesenchymal stem cells. 5. The encapsulated microsphere of any one of the preceding embodiments, wherein the encapsulated microsphere comprises 2 to about 1x106chondrocytes. 6. The encapsulated microsphere of any one of the preceding embodiments, wherein the encapsulated microsphere has a diameter of about 1 ^m to about 1 mm. 7. The encapsulated microsphere of any one of the preceding embodiments, wherein the encapsulated microsphere has a diameter of about 100 ^m to about 700 ^m. 8. The encapsulated microsphere of embodiment 7, wherein the encapsulated microsphere has a diameter of greater than about 400 ^m to less than about 650 ^m. 9. The encapsulated microsphere of any one of the preceding embodiments, wherein the clusters of cells have an average equivalent circular diameter of about 20 ^m to about 200 ^m. 10. The encapsulated microsphere of any one of the preceding embodiments, wherein the cells in the encapsulated microsphere further comprise an exogenous polynucleotide. 11. The encapsulated microsphere of embodiment 10, wherein the exogenous polynucleotide comprises a sequence encoding a polypeptide.MGH 2024-400-02 Quarles ref.125141.04818 The encapsulated microsphere of embodiment 11, wherein the polypeptide comprises a nucleic acid-guided nuclease. The encapsulated microsphere of embodiment 12, wherein the nucleic acid-guided nuclease comprises a Cas nuclease. The encapsulated microsphere of embodiment 13, wherein the Cas nuclease comprises Cas9. The encapsulated microsphere of any one of embodiments 12-14, wherein the encapsulated microsphere further comprises a sequence encoding a guide RNA (gRNA). A tissue construct comprising a plurality of the encapsulated microspheres of any one of embodiments 1-15. The tissue construct of embodiment 16, wherein the plurality of encapsulated microspheres is 3 to 1000 encapsulated microspheres. The tissue construct of embodiment 17, wherein the tissue construct is about 1 mm to about 10 mm in diameter. The tissue construct of embodiment 18, wherein the tissue construct is about 9 mm in diameter. The tissue construct of any one of embodiments 16-19, wherein the tissue construct comprises smooth white surfaces and / or glycosaminoglycan deposition throughout the construct, optionally, as detected by alcian blue staining. An orthopedic graft comprising a plurality of the encapsulated microspheres of any one of embodiments 1-15 affixed to cartilage, a bone, or a bone fragment. The orthopedic graft of embodiment 21, wherein the plurality of encapsulated microspheres are affixed to the bone or the bone fragment in a matrix comprising fibrin. The orthopedic graft of embodiment 22, wherein the matrix further comprises thrombin or collagen type I. The orthopedic graft of any one of embodiments 21-23, wherein the bone or bone fragment is decellularized.MGH 2024-400-02 Quarles ref.125141.04818 A method of generating encapsulated microspheres, the method comprising, at an apparatus comprising a first dispensing unit comprising a first nozzle and a second dispensing unit comprising a second nozzle, (a) forming a first droplet at the first nozzle comprising at least one cell and a first matrix and forming a second droplet at the second nozzle comprising a second matrix; (b) dispensing the first droplet onto a substrate and dispensing the second droplet onto the substrate such that the first droplet and the second droplet collide to form an encapsulated microsphere, wherein the second droplet is dispensed at a velocity sufficient to disrupt the arrangement of cells in the first droplet, thereby generating clusters of cells within the encapsulated microsphere, and wherein the first matrix and the second matrix comprise reagents that form a gel or a solid following the collision of the first droplet and the second droplet. The method of embodiment 25, wherein the first matrix comprises fibrin and the second matrix comprises thrombin or the first matrix comprises thrombin and the second matrix comprises fibrin. The method of embodiment 25 or 26, wherein the first matrix comprises fibrin and the second matrix comprises thrombin. The method of any one of embodiments 25-27, wherein the first droplet is dispensed such that the cells achieve a relatively homogenous distribution in the droplet. The method of any one of embodiments 25-28, wherein there is no interface between the clusters of cells within the encapsulated microsphere. The method of any one of embodiments 25-29, wherein at least one cell comprises a chondrocyte. The method of any one of embodiments 25-30, wherein the concentration of the cells in the first droplet is about 6x106cells / ml to about 10x106cells / ml. The method of any one of embodiments 25-31, wherein the concentration of the fibrin is about 2 mg / ml.MGH 2024-400-02 Quarles ref.125141.04818 The method of any one of embodiments 25-32, wherein the concentration of thrombin is about 1 unit / ml to about 2 units / ml. The method of any one of embodiments 25-33, wherein either the first of the second matrix comprises collagen type I. The method of embodiment 34, wherein the concentration of collagen type I is about 2 mg / l. The method of any one of embodiments 25-35, wherein the first and second dispensing unit each comprise a syringe including (i) a hollow body and a plunger dimensioned to translate in the body, the body having an exit orifice; (ii) an actuator in contact with a proximal end of the plunger; (iii) a controller for controlling linear motion of the actuator wherein the controller executes a program received from a computer in the controller to drive the actuator toward the proximal end of the plunger to dispense a bioink from the body of the syringe; and (iv) a nozzle having a wall defining a fluid path extending from an inlet of the nozzle to an outlet of the nozzle, the inlet of the nozzle being in fluid communication with the exit orifice of the body of the syringe, wherein as the controller drives the actuator toward the proximal end of the plunger bioink flows into the fluid path, wherein the nozzle includes a fluid passageway in fluid communication with a source of fluid and the fluid path, wherein the source of fluid comprises a controllable valve for supplying pulsed fluid from the source of fluid to the fluid passageway to create separate droplets from the flow of bioink in the fluid path in the nozzle by blowing the droplets away from the nozzle using the pulsed fluid from the source of fluid. The method of any one of embodiments 25-36, wherein the dispensing comprises blowing the droplets away from the nozzle using pulsed fluid from the source of fluid. The method of embodiment 37, wherein the pulsed fluid comprises a gas.MGH 2024-400-02 Quarles ref.125141.04818 The method of embodiment 38, wherein the pulsed fluid comprises pulsed air. The method of embodiment 39, wherein the air comprises millisecond pulsed air. The method of any one of embodiments 25-40, wherein a volume of each droplet is in a range of about 10 nanoliters to about 15 microliters. The method of any one of embodiments 25-41, wherein the method further comprises repeating steps (a) and (b) n additional times, wherein n is an integer greater than 1, to generate n encapsulated microspheres. The method of any one of embodiments 25-42, wherein the first droplet comprises 2 to about 1x106chondrocytes. The method of any one of embodiments 25-43, wherein the encapsulated microspheres have a diameter of about 1 ^m to about 1 mm. The method of embodiment 44, wherein the encapsulated microspheres have a diameter of about 100 ^m to about 700 ^m. The method of embodiment 45, wherein the encapsulated microspheres have a diameter of greater than about 400 ^m to less than about 650 ^m. The method of embodiment 42, wherein the n encapsulated microspheres are arranged on the substrate to generate a tissue construct precursor. The method of any one of embodiments 25-46, wherein the substrate is a partial-thickness defect in articular cartilage. The method of any one of embodiments 25-46, wherein the substrate is a full-thickness defect in articular cartilage. The method of embodiment 47, wherein the tissue construct precursor is further cultured in vitro for about 1 day to about 10 weeks to generate a tissue construct. The method of embodiment 42, wherein the method further comprises applying the n encapsulated microspheres to a cartilage defect. The method of embodiment 51, wherein applying comprises depositing the n encapsulated microspheres in a delivery matrix.MGH 2024-400-02 Quarles ref.125141.04818 The method of embodiment 52, wherein the delivery matrix comprises fibrin. The method of embodiment 53, wherein the delivery matrix further comprises collagen type I. The method of any one of embodiments 52-54, wherein the method further comprises applying a solidifying matrix comprising thrombin to the delivery matrix to generate a solidified matrix comprising the encapsulated microspheres. The method of any one of embodiments 25-55, wherein the method further comprises repeating steps (a) and (b) n times and varying at least one parameter of encapsulated microsphere formation in each of the n repeats, to generate n encapsulated microspheres with varying properties; analyzing at least one parameter related to cartilagenesis in each of the n encapsulated microspheres; and selecting at least one parameter of encapsulated microsphere formation, based on the analysis of the at least one parameter related to cartilagenesis. The method of embodiment 56, wherein the at least one parameter of encapsulated microsphere formation comprises pulsed fluid pressure of the first and or second nozzle. The method of embodiment 56 or 57, wherein the at least one parameter related to cartilagenesis comprises equivalent circular diameter of the clusters in the encapsulated droplet or expression of at least one gene or protein. The method of embodiment 58, wherein the at least one gene or protein comprises collagen type II (COL-II), transcription factor SOX-9 (SOX9), aggrecan (ACAN), or collagen type I (COL-I). The method of embodiment 59, wherein at COL-II, SOX9, and ACAN are positively related to cartilagenesis and COL-I is negatively related to cartilagenesis. A method of testing a candidate therapeutic, the method comprising contacting the candidate therapeutic to at least one of the encapsulated microspheres of any one of embodiments 1-15.MGH 2024-400-02 Quarles ref.125141.04818 62. The method of embodiment 61, wherein the method further comprises detecting at least one parameter associated with performance of the candidate therapeutic. 63. The method of embodiment 62, wherein detecting the at least one parameter comprises detecting gene expression, protein expression, glycosaminoglycan (GAG) deposition, or protein secretion. 64. The method of any one of embodiments 61-63, wherein the method further comprises contacting the at least one encapsulated microsphere with an inflammatory reagent. 65. The method of embodiment 64, wherein the inflammatory reagent comprises interleukin 1 beta (IL-1^). EXAMPLES Example 1 - Cellular Patterning Alone Using Bioprinting Regenerates Articular Cartilage Through Native-Like Cartilagenesis

[0093] Example 1 is based on Grottkau, Brian E., et al., “Cellular Patterning Alone Using Bioprinting Regenerates Articular Cartilage Through Native-Like Cartilagenesis” Small. 2024 Aug;20(31):e2308694, which is authored by the inventor and incorporated by reference herein in its entirety.

[0094] Few studies have proved that bioprinting itself helps recapitulate native tissue functions mainly because the bioprinted macro shape can rarely, if ever, influence cell function. This can be more problematic in bioprinting cartilage, generally considered more challenging to engineer. Here a new method is shown to micro-pattern chondrocytes within bioprinted sub-millimeter micro tissues, denoted as patterned micro-articular-cartilages tissues (PA-MCTs). Under the sole influence of bioprinted cellular patterns. A pattern scoring system is developed after over 600 bioprinted cellular patterns are analyzed. The top-scored pattern mimics that of the isogenous group in native articular cartilage. Under the sole influence of this pattern during PA-MCTs bio- assembling into macro-cartilage and repairing cartilage defects, chondrogenic cell phenotype is preserved, and cartilagenesis is initiated and maintained. Neocartilage tissues from individual and assembled PA-MCTs are comparable to native articular cartilage and superior to cartilage bioprinted with homogeneously distributed cells in morphology, biochemical components,MGH 2024-400-02 Quarles ref.125141.04818 cartilage-specific protein and gene expression, mechanical properties, integration with host tissues, zonation forming and stem cell chondrogenesis. PA-MCTs can also be used as osteoarthritic and healthy cartilage models for therapeutic drug screening and cartilage development studies. This cellular patterning technique can pave a new way for bioprinting to recapitulate native tissue functions via tissue genesis.

[0095] Introduction

[0096] Live-cell 3D bioprinting has recently progressed considerably. This holds promise in solving organ shortages and in providing better models to predict drug treatment outcomes prior to clinical trials. However, few studies have proved that bioprinting itself has helped recapitulate native tissue functions. Unlike a 3D-printed construct using only polymers, a bioprinted construct using live cells must go through a post bioprinting process to mature. To positively influence a bioprinted construct to mature toward its native analog tissue, bioprinting itself needs to provide a cue to initiate and maintain tissue genesis during the post-bioprinting culture. However, existing bioprinting techniques largely fail to accomplish this goal.

[0097] Numerous reported successes have focused on generating anatomical tissue shapes at macro-scales. However, tissue functions are determined by cells, which are largely unable to sense the influence of a macro-scale shape. In contrast, as proved by other bioengineering techniques, a micro-scale cellular pattern is an important biophysical signal in the extracellular microenvironment. Cellular patterns determine cell fates and phenotypes and ultimately determine tissue genesis and functions.

[0098] Unfortunately, existing bioprinting techniques are not capable of patterning cells at a micro-scale to provide this favorable cue. Even with the highest cellular resolution, single-cell droplet bioprinting is not suitable for this patterning purpose because it cannot print any hydrogel. Although existing hydrogel-containing droplet-based techniques bioprint the smallest unit, these droplets almost all spread and merge with neighboring droplets resulting in the cells in the entire construct being homogeneous (FIG. 1A, FIG. 1B). If individual droplets are solidified upon reaching a substrate, matrix interfaces form between droplets. This interface separation limits matrix remodeling and inhibits the bioprinted tissue from maturing (FIG.1C).

[0099] We have recently developed a new bioprinting technique—directly controlled droplet bioprinting (DVDOD). Using DVDOD, we can pattern cells into multiple clusters within a dropletMGH 2024-400-02 Quarles ref.125141.04818 of bioink. Using optimized bioprinting parameters, we can control the distribution patterns of cell clusters and the number and area percentages of the clusters that support chondrogenesis and cartilagenesis. Here, we report the success of generating native-comparable sub-millimeter patterned micro-articular-cartilages tissues (PA-MCTs).

[0100] Articular cartilage has a unique pattern of isogenous groups. Articular cartilage defects are prevalent, and osteoarthritis (OA) is the leading cause of disability. Because of donor shortage, native-compatible engineered cartilage is highly desired for clinical, pharmaceutical, and research applications. Articular cartilage is composed of only one cell type without any vasculature or lymphatic vessels and was predicted to be one of the first tissues to be successfully engineered. However, numerous studies over 30 years have shown that the simplicity of the cellular component and the complexity of the structural organization of cartilage makes it more difficult to engineer than other cellularly complex tissues. The main reason for this difficulty is that chondrocytes lose their native phenotype and cartilagenesis rarely happens during the in vitro process. The intricate pattern observed in articular cartilage originates from tightly grouped clusters of chondrocytes that form during the prenatal development phase known as cartilagenesis. This specific arrangement endures throughout the entire lifespan of healthy cartilage tissue. Because of this, we postulate that patterning chondrocytes as clusters alone, without the need for exogenous factors, is sufficient to preserve the function of native chondrocytes and to both initiate and sustain the process of cartilagenesis.

[0101] Here we demonstrate that without any exogenous factors, solely under the cue of the bioprinted cellular pattern, the native phenotype of chondrocytes is preserved and the chondrogenesis of human mesenchymal stem cells is enhanced. The bioprinted pattern also initiates and maintains cartilagenesis in individual PA-MCTs and during bio-assembly (PA-MCTs integration). PA-MCTs are comparable to native articular cartilage in morphology, biochemical components, cartilage-specific protein and gene expression, and mechanical properties. PA-MCTs can also be used as miniature models of healthy and osteoarthritic (OA) cartilage. MCTs bioprinted with homogeneously distributed cells (H-MCTs) were used as the control.

[0102] Results

[0103] In our study, we successfully demonstrate the application of the DVDOD technique for patterning cells. This method transforms a uniform distribution of cells into distinct clusters withinMGH 2024-400-02 Quarles ref.125141.04818 the smallest unit of bioprinting: a single droplet. DVDOD operates in two key steps: i) a predetermined volume of bioink is precisely moved to the nozzle tip through a linear motion mechanism in the syringe plunger, and ii) the droplet is then accurately ejected onto the substrate using controlled, pulsed air. A unique aspect of DVDOD is that the volume of the dispensed bio- ink droplet remains constant, regardless of the air pressure used, as shown in FIGS.2A-2F.

[0104] Utilizing this feature, DVDOD allows for the collision of different bioink droplets, leading to the formation of cellular clusters within a single droplet. This is achieved using minimal pressure to dispense the bioink. Subsequently, a second layer of bioink, containing thrombin but no cells, is deposited. The interaction of this second layer with the first transforms the uniform chondrocyte distribution into clusters. The thrombin quickly solidifies the fibrin, encapsulating the droplet and thus fixing the chondrocyte clusters in place without any interfaces between them. The process and the resultant patterns are illustrated in FIG.2A-ii and FIG.2E-i.

[0105] We used the distributions of the equivalent circular diameter (ECD) of cell clusters to represent the patterns of cell clusters within a bioprinted micro-construct (deliberately controlling the ID >2 mm to include more cell clusters for analysis, FIG.2B-ii, FIG.2E-i,ii). By varying the volumes of droplets, driving pressures, the number of droplets, and the location of droplet- dispensing, we generated different patterns of cell clusters (FIG. 2B-i). Using Empirical Cumulative Distribution Function (ECDF, FIG.2C) plotting and Kolmogorov-Smirnov (KS, FIG. 2D) distribution analysis, we selected 20 unique patterns from an array composed of over 600 patterns as the standard patterns—every two patterns are statistically different (FIG.2B-iii, FIG. 2D). Replicates of each standard pattern were bioprinted using the same corresponding bioprinter control parameters. Only the replicates with the statistically same distribution as the corresponding standard distribution were included for further pattern analysis (FIG.2B-iv; FIGS.3A-3C). Within the same assay, using the same lot of cells and hydrogels, 20 groups of PA-MCTs of 20 same unique patterns were also bioprinted using the corresponding bioprinter control parameters. The PA-MCTs were further cultured for 3 weeks before qPCR analysis of the expression of the cartilagenesis positively related genes Col-II, Sox-9 and ACAN and the negatively related gene COL-I (FIG.2B-v, FIG.2F).

[0106] One of our goals was to develop a scoring system to quickly select the pattern that best supports cartilagenesis during each bioprinting assay's initial bioprinter calibration stage. UsingMGH 2024-400-02 Quarles ref.125141.04818 the bioprinter control parameters among the 20 unique patterns, we selected the optimized pattern supporting chondrogenesis based on qPCR scores (FIG.2F). Because KS distribution analysis is nonparametric and it does not rank multiple distributions, we chose three parameters to represent the cluster patterns: i) Area percentage: the summed area of clusters in a specific ECD range over the total cluster area, which reflects the percentage of cells under the influence the pattern (bar plots in FIG.4A-ii) Number percentage: the summed number of clusters in a specific ECD range over the total cluster numbers, which reflects the efficiency of patterning (raincloud plots in FIG. 4A-iii) Distances between clusters (FIG.2E-iii). First, we observed that once the distances between clusters were greater than 15 µm, which was observed in most of the selected patterns, the cartilagenesis was not affected. Therefore, this parameter was excluded from the scoring system. We introduced a pattern score (FIG. 2B-iv), the average of the number percentage and area percentage of the clusters in a specific ECD range. We chose 20 µm as the lower limit of the ECD range because clusters with ECD ≤ 20 µm can form spontaneously, and 220 µm as the upper limit to exclude samples containing large homogenous clusters. We evaluated pattern scores calculated from 66 different ECD ranges (Table 1, Supporting Information), and ranked each pattern and qPCR score in descending order (FIG.4B-i).

[0107] Table 1.66 ECD ranges for the quantification of cluster distributions. (1) 20-50 µm (2) 20-60 µm (3) 20-70 µm (4) 20-80 µm (5) 20-90 µm (6) 20-100 µm (7) 20-110 µm (8) 20-120 µm (9) 20-130 µm (10) 20-140 µm (11) 20-150 µm (12) 30-60 µm (13) 30-70 µm (14) 30-80 µm (15) 30-90 µm (16) 30-100 µm (17) 30-110 µm (18) 30-120 µm (19) 30-130 µm (20) 30-140 µm (21) 30-150 µm (22) 40-70 µm (23) 40-80 µm (24) 40-90 µm (25) 40-100 µm (26) 40-110 µm (27) 40-120 µm (28) 40-130 µm (29) 40-140 µm (30) 40-150 µm (31) 50-80 µm (32) 50-90 µm (33) 50-100 µm (34) 50-110 µm (35) 50-120 µm (36) 50-130 µm (37) 50-140 µm (38) 50-150 µm (39) 60-90 µm (40) 60-100 µm (41) 60-110 µm (42) 60-120 µm (43) 60-130 µm (44) 60-140 µm (45) 60-150 µm (46) 70-100 µm (47) 70-110 µm (48) 70-120 µm (49) 70-130 µm (50) 70-140 µm (51) 70-150 µm (52) 80-110 µm (53) 80-120 µm (54) 80-130 µm (55) 80-140 µm (56) 80-150 µm (57) 90-120 µm (58) 90-130 µm (59) 90-140 µm (60) 90-150 µm (61) 100-130 µm (62) 100-140 µm (63) 100-150 µm (64) 110-140 µm (65) 110-150 µm (66) 120-150 µmMGH 2024-400-02 Quarles ref.125141.04818 ECD: equivalent circle diameter

[0108] According to Spearman's correlation analysis of the ranking (FIG. 2B-vi), the pattern scores using the ECD range of 20–80 µm have the highest correlation with the corresponding qPCR scores (FIG.4B-ii). This means that when evaluated using the 20–80 µm ECD range, the pattern of the highest score ranking best supports cartilagenesis. For each assay, after an array of micro-constructs is bioprinted using different printer control parameters, pattern scores were calculated. The control parameters with the highest score were selected for each bioprinting batch. H-MCTs were bioprinted without generating cell clusters by omitting the droplet colliding. The final concentrations of cells and hydrogels in PA-MCTs and H-MCTs and the volumes of corresponding PA-MCTs and H-MCTs were controlled to be the same in each assay.

[0109] The conceptual design of assays used to analyze the PA-MCTs is illustrated in FIG.5. First, cluster-patterning alone in PA-MCTs preserved the native phenotype of chondrocytes as evidenced by morphology, gene expression, and mechanical properties. Compared with clustered cellular patterns in PA-MCTs, H-MCTs showed homogeneous chondrocyte distribution after bioprinting (FIG.6B-i). The overall sizes of the individual PA-MCTs are uniform (FIG.6B-ii). Our bioprinting process does not affect cell viability (FIG. 6B-iii). Chondrocytes in PA-MCTs also expressed significantly higher levels of native phenotype (chondrogenic) genes, namely, collagen type II and aggrecan, than those in H-MCTs, and a significantly lower level of the fibrotic phenotype gene of collagen type I at week 5 (FIG.6D).

[0110] At the tissue level, cluster patterning also initiates and maintains the cartilagenesis (FIG. 6A) in PA- MCTs as evidenced by morphology and biochemical components, biomechanical enhancement, and overall scores. PA-MCTs show smooth white surfaces, which is a typical macroscopic appearance of native cartilage (FIG.6E-i). PA-MCTs are rigid and small enough to be delivered through injection (FIG. 6E-ii). Microscopically, lacunae and isogenous groups, the structural morphology of native cartilage, formed at week 3 and became extensive by week 5 (FIG. 6C-I and FIG. 7). In contrast, lacunae or isogenous groups did not form in H-MCTs, similar to engineered cartilage reported previously using homogeneous cells (FIG. 6C-i). For biochemical components, i) collagen type II content was significantly higher in PA-MCTs than in H-MCTs and showed no difference compared with native cartilage (FIG. 6C-ii,G); ii) sulfatedMGH 2024-400-02 Quarles ref.125141.04818 glycosaminoglycans (sGAG) content was higher in PA-MCTs than in native cartilage and significantly higher than in H-MCTs (FIG.6C-i,F). For biomechanical properties, as characterized by a capillary aspiration assay (FIG. 6H-i), PA-MCTs also demonstrated significantly greater stiffness than H-MCTs (FIG.6H-ii). PA-MCTs also demonstrated a significantly higher Modified Bern Score—an overall evaluation of the quality of tissue-engineered cartilage—than H-MCTs (FIG.8, Table 3).

[0111] Previously, modular cartilage tissues, including chondrocyte spheroids or miniature cubes of minced native cartilage, were expected to assemble into a macro-cartilage-like tissue through a bottom-up approach. However, because chondrocytes change to fibrotic phenotype in the spheroids and cartilagenesis is absent, spheroids are only able to fuse into a small and thin (<1.5 mm in long axis, <0.75 mm in height) cartilage-like tissue. A larger fusion usually assembles incompletely in vitro: boundaries of individual modules remain; necrotic cores form; and mechanical properties of fused tissues are poor. For minced cartilage, cartilage-to-cartilage integration is extremely difficult to accomplish because low metabolism and dense anti-adhesive matrix exist in native cartilage and cartilagenesis cannot be initiated.

[0112] In contrast, our approach of bioprinted cellular patterning solved both problems mentioned above. Without any exogenous factors, the biophysical cue of cellular patterning maintains the native phenotype of chondrocytes. After an initial short period of cartilagenesis (2 weeks) in individual PA-MCTs, the cartilagenesis continues during the entire bio-assembly process. Thus, we successfully generated macro-articular-cartilage tissues.

[0113] First, using less than 20 PA-MCTs, we studied the dynamics of cartilagenesis during bio- assembly using the process illustrated in FIG.9A using a simple model. Bio-assembly, as we are using the term, refers to the integration of micro-tissues (building blocks or modular tissues) to form macro-cartilage as opposed to self-assembly, where cells attach to form a spheroid or a sheet (a single building block). The goal of the simple model is to investigate the morphology dynamic. We found three major changes occurred and the activity ratios between any two changes are dynamic over the time course (FIG. 9C, FIGS. 10A-10B): i) Cell migration. Chondrocytes migrated out of PA-MCTs into the surrounding hydrogel. This occurred as early as the first 24 h when a single PA-MCT was tracked. The Chondrocyte phenotype was well preserved, as indicated by positive Alcian blue staining (FIG. 9B). ii) New matrix deposition. The chondrocytes thatMGH 2024-400-02 Quarles ref.125141.04818 migrated to the spaces between PA-MCTs deposited sGAG starting from week 1 (FIG.9C; FIGS. 11A-11C, Supporting Information). The newly deposited matrix interconnected individual PA- MCTs. iii) Structure reorganization (FIG. 9C). Each PA-MCT began reorganizing internal structures as the boundary of each PA-MCT gradually diminished starting from week 5. When reorganization was completed at week 7, individual PA-MCTs disappeared, and the macro-tissue showed a global native-articular cartilage morphology.

[0114] We further demonstrate that the biophysical cue of bioprinted cellular patterns can maintain cartilagenesis when ≈800 PA-MCTs are assembled into large macro-cartilage at a clinically- applicable scale (9 mm in diameter). Cartilagenesis was evidenced by native-comparable morphology, as well as matrix components including GAG and type II collagen, and enhanced mechanical properties. Macroscopically (FIG.9D), when PA-MCTs were loaded into a transwell and covered with hydrogels, individual PA-MCTs were clearly visualized. At week 5, PA-MCTs were bio-assembled into a macro-tissue and its surface became flat, and only blurry outlines of the individual PA-MCTs were visualized. At week 7, the macro-tissue showed articular cartilage- comparable appearances: smooth white surfaces; and intense sGAG deposition as evidenced by dark blue throughout the tissues after whole-tissue Alcian blue staining (FIG. 9E). At the microscopic level, whole-mount Safranin O staining (FIG. 9F, FIG. 12) shows that sGAG was intensely expressed throughout the macro-tissue. All the PA-MCTs were interconnected and the assembly was structurally seamless. No necrotic morphology was observed in any space within the assembled cartilage. Collagen type II was also intensely expressed in assembled macro- articular-cartilage tissues (FIG.9F-i). In comparison, similar to the results previously reported by others, H-MCTs did not fully assemble and gaps were visualized both macroscopically and microscopically (FIG. 9F-ii) at week 7. H-MCTs did not show native-cartilage-like morphology, and the assembled macro-tissues showed significantly less intense sGAG staining and fewer cells. The chondrocytes in the H-MCTs showed elongated fibrotic morphology and also behaved like fibroblasts by compacting individual H-MCTs into irregular shapes (FIG. 9F-ii). The assembled macro-articular-cartilage tissues from PA-MCTs demonstrated superior mechanical properties to those from H-MCTs (FIG. 9G): i) significantly higher Young's modulus, ii) significantly higher aggregate modulus, and iii) significantly lower permeability constant indicating the PA-MCTs formed more mature native-like—more solid and less porous—internal structures.MGH 2024-400-02 Quarles ref.125141.04818

[0115] Next, we demonstrate that PA-MCT-assemblies are able to integrate with host cartilage and subchondral bone through cartilagenesis. As mentioned above, native cartilage-to-cartilage integration rarely happens. Therefore, clinically, the capability of an engineered implant to integrate with host cartilage and bone determines its success. In the integration assay, macroscopically, PA-MCT-assemblies grew into smooth white macro-cartilage on top of the native cartilage and bone (FIG.13-i). Microscopically, the PA-MCT-assemblies formed articular cartilage and showed seamless histological integration with the host cartilage and bone tissues (FIG.13-iii). In contrast, at week 7 of in vitro culture, H-MCT-assemblies failed to form intact macro-cartilage as individual H-MCTs were still visible macroscopically (FIG. 13-ii), and void spaces were visualized throughout the samples microscopically (FIG.13-iv). The H-MCT-assemblies failed to attach to the native cartilage as a sizable gap was observed between individual H-MCTs (FIG.13- ii top). Most H-MCT-assemblies fell off while a few loosely attached to the underlying bone (FIG. 13-ii bottom). Intense matrix expression of sGAG (FIG. 13-iii) was observed in the PA-MCT- assemblies. The sGAG content of PA-MCT-assemblies in the cartilage and bone integration assays were 5.4-fold (p < 0.01) and 6.9-fold (p < 0.05) of those in H-MCT-assemblies, respectively (FIG. 14).

[0116] We further demonstrate the capability of PA-MCTs to repair cartilage defects through cartilagenesis in a more clinically relevant model (FIG. 15A). Two types of articular cartilage defects exist clinically: i) a partial-thickness defect, which is difficult to treat because existing implants do not adhere to it, and ii) a full-thickness defect, which is a more advanced stage of the disease. We demonstrate repairing both per bovine osteochondral allografts. Mimicking a clinical minimally invasive approach, PA-MCTs were injected into the defect. The outer diameter of the delivery device is small enough (<4 mm) to potentially operate in a minimally invasive manner during surgery (FIG.6E-ii; FIGS.16A-16D).

[0117] Macroscopically (FIG. 15B): i) at weeks 2 and 5, individual PA-MCTs had partially assembled, and their outlines became blurry; ii) at week 7, PA-MCTs assembled into macro- articular-cartilage with a smooth white surface. The macro-articular-cartilage repaired the two types of defects completely by filling the full-thickness defect and by adding a layer of cartilage tissue to the partial-thickness defect, increasing its height to normal. Microscopically, PA-MCT- assembly seamlessly integrated with the irregularly shaped cartilage walls (FIG.15C-i), the host subchondral bone (FIG.15C-ii), and the partial-thickness defect (FIG.15C-iii), demonstrating theMGH 2024-400-02 Quarles ref.125141.04818 native-comparable morphology. Intense sGAG was deposited throughout the PA-MCT-assembly, including the deep zone (FIG. 15C-iv), as demonstrated by the color intensity quantification of Safranin O staining (FIG.15F). As the depth of the defect that PA-MCTs repaired (3 mm) is greater than the average depth of cartilage in the human knee (2.14 mm), PA-MCTs show promise for clinical application. In comparison, H-MCTs failed to repair defects, which is similar to the result in our integration assay and those reported elsewhere using spheroids composed of homogeneously distributed chondrocytes. A large proportion of the defect was void due to unassembled H-MCTs that fell off the explants during culture and a large degree of H-MCT compaction (FIG. 15D). Histologically, necrosis, compaction of H-MCTs, void space underneath the neo-tissue surface, and unconnected and unintegrated H-MCTs were observed (FIG. 15E). The average adhesion strength of PA-MCT-assemblies to the host cartilage reached 26% of the force needed to push to tear an intact healthy native cartilage tissue. On average, PA-MCT-assemblies showed a 9.6-fold higher adhesion strength than H-MCT-assemblies (FIG.15H and p < 0.05). Explants repaired by PA-MCTs also demonstrated a significantly higher Modified O'Driscoll Score—an overall evaluation of the quality of cartilage defect repair—than those by H-MCTs (FIG.17, Table 4).

[0118] The unique zonal structure developed during postnatal cartilagenesis largely improves the capability of native articular cartilage to withstand shear and compressive forces. However, previous attempts, including 3D bioprinting to create articular cartilage zonation, have not been successful. These attempts mostly construct a layered structure using different materials or concentrations of cells. However, the initially layered setup does not lead to growing a zonal structure at the end of the assay. Instead, we rely on the capability of cartilagenesis to create the zonation. We observed encouraging zonal structure formed in the macro-articular-cartilage assembled from PA-MCTs, but not from H-MCTs (FIG.15G). Three structure zones can be clearly visualized: i) the top zone is composed of flat and elongated chondrocytes being parallel to the cartilage surface, which almost recapitulates the characteristics of the superficial zone in native articular cartilage; ii) some chondrocyte-stacks, perpendicular to the cartilage surface, formed in the bottom zone. This represents some characteristics of the deep zone in native articular cartilage; iii) the medial zone, corresponding to the native intermediate zone, is distinct from the top and bottom zones by its morphology but resembles the native characteristics less than the other two zones do. Interestingly, when PA-MCTs were assembled without integrating into a native tissue (FIG.9F), the zonal structure did not form. We think the possible factors determining this mightMGH 2024-400-02 Quarles ref.125141.04818 be some cues from the native tissue and / or the asymmetry between the native tissue and the culture medium. Future study is needed to elucidate this and other articular cartilage zonal properties, such as biomechanical and biochemical zonations. The formation of zonal structure is another evidence that cartilagenesis occurs during the assembly process.

[0119] We also demonstrated the feasibility of several potential therapeutic applications using PA- MCTs and analyzed their cartilagenesis capability in a proof-of-concept manner. i) Repairing large (long axis > 15 mm) irregularly-shaped cartilage defects (FIG.15I): it was modeled in clinically relevant sizes for a translational purpose; in addition, PA-MCTs can self-fit concave and convex surfaces, demonstrating the potential to be applied in complex-shaped defects (FIGS.18A-18B). ii) Resurfacing entire femoral condyles (FIG. 15J): this demonstrates the potential of cartilage regeneration for the whole joint, iii) Assembling and generating a personalized osteochondral graft from the data of a patient's computed tomography: it can be potentially used to treat a more complex articular disease of an osteochondral defect where both cartilage and bone losses have occurred. (FIGS.19A-19M) and other information detailed in the Supporting Information.

[0120] In addition to the characteristics for therapeutic applications that we have demonstrated above, PA-MCTs can also be used as miniature models in drug screening and developmental biology studies. Miniature models are largely preferable because of their higher throughput. However, existing spheroid models using homogeneously distributed chondrocytes poorly represent native cartilage. A native cartilage explant (typically 3 mm in diameter, 1 mm in height) is too large (more than 60 times the volume of a PA-MCT) to be used for drug screening. Additionally, explants have topography-related variations even when harvested from the same joint. In contrast, as evaluated by the coefficient of variation (CV) of sGAG content, PA-MCTs demonstrated a smaller variation than all groups of explants and significantly smaller variation than explants from the medial tibial plateau, lateral femoral condyle, and the entire joint (FIG. 20A). Aiming at disease-modifying osteoarthritis drugs (DMOADs), we developed a micro- osteoarthritis model (OA-PA-MCTs) using PA-MCTs, and it demonstrated native arthritis-like characteristics (FIG.20B). Using OA-PA-MCTs, we tested several biomolecules with known anti- inflammatory effects. Compared to native articular cartilage reported previously, OA-PA-MCTs responded to these drugs similarly (FIG.20C): Dexamethasone, IGF-1 and TGF-β demonstrated robust effects in reducing sGAG loss, inhibited expression of inflammatory genes (MMP-3 and MMP-13) and up-regulated expression of cartilage positively related genes (Sox9, collagen typeMGH 2024-400-02 Quarles ref.125141.04818 II and ACAN) than Celecoxib. The overall anti-inflammatory ranking of drugs is in the order: Dexamethasone > IGF-1 > TGF-β > Celecoxib (Table 2).

[0121] Table 2. Ranking of the disease-modifying effects of the drugs

[0122] Ranking ScoreIL-1β+DEX IL-1β+IGF-1 IL-1β+TGFβ IL-1β+CELCartilage-Positive-Genes 2 3 4 1 Anti Inflammation-Genes 4 3 2 1 GAG Content 4 3 2 1 Overall 10 9 8 3 DEX: Dexamethasone, CEL: Celecoxib

[0123] Collectively, OA-PA-MCTs show native-OA-like characteristics with high uniformity and miniature size, which make them suitable for DMOADs screening. PA-MCTs also show an advantage in studies of cartilage development biology. Microinjecting genes into a cartilage explant is superior to transfecting genes into individual chondrocytes because it models at a tissue or organ level to study related signal pathways in normal cartilage development. However, this method suffers the same limitation of explant variations. We demonstrate that PA-MCTs can overcome this limitation by successfully microinjecting dextran, which mimics a gene carrier such as adenovirus, into PA-MCTs as a proof-of-concept study (FIG. 20D). Similar to our drug screening model, PA-MCTs can increase throughput and minimize sample variations compared with native cartilage explants.

[0124] Lastly, in a preliminary attempt, PA-MCTs were bioprinted with hMSCs (hMSC-PA- MCTs) using the same bioprinting approach. We demonstrate that cellular patterning (FIG.20E) also enhances chondrogenic differentiation of hMSCs and initiates and maintains cartilagenesis in individual hMSC-PA-MCTs and their assemblies. hMSC-PA-MCTs also showed the native- cartilage-like characteristics in morphology (FIG.20E), sGAG content (FIG.20F), chondrogenic gene expression (FIG.20G), and capability of bio-assembly (FIG.20H). All these characteristics are superior to those of hMSCs-H-MCTs (bioprinted with homogeneous hMSCs). Specifically, similar to fibrotic cell morphology observed in our H-MCTs and in hMSC-spheroids reported by others without a biophysical cue, differentiated cells in hMSCs-H-MCTs showed fibroblast-likeMGH 2024-400-02 Quarles ref.125141.04818 characteristics as well (FIG. 20E). Again, we prove that a bioprinted cellular pattern itself can regulate the function of chondrogenic cells, including both hMSCs and chondrocytes and also regulate cartilagenesis.

[0125] Conclusion

[0126] In summary, to the best of our knowledge, this is the first time that patterning cells within a bioprinted unit has been accomplished and its practical significance demonstrated. We report the success of recapitulating native functions and characteristics of articular cartilage in PA-MCTs and their assemblies solely by bioprinted cellular patterns. The patterned structure provides a biophysical cue that positively influences cells toward the chondrogenic phenotype and initiates and maintains cartilagenesis. Our technique represents a potential solution to arthritis. As this study serves as a proof of concept, future investigations into the molecular mechanisms underlying these processes, including comprehensive gene analysis, will be beneficial. We anticipate future research in implanting PA-MCTs in vivo, screening new candidate DMOADs, and generating other modular tissues through bioprinted cellular patterns to activate the corresponding tissue genesis. This technique could pave a new avenue for bioprinting to recapitulate native tissue functions.

[0127] Supporting Information

[0128] 3D Bioprinting

[0129] The Bioprinter

[0130] The details of the direct-volumetric drop-on-demand (DVDOD) 3D bioprinter have been reported previously. In our system, the bioink droplet volume is directly regulated by a linear actuator, a departure from the indirect control mechanisms observed in existing systems, such as modulating fluid-valve opening durations or the pressure applied to the bioink. The bioprinter, in essence, comprises a three-axis linear motion gantry outfitted with multiple linear dispensing units. Each unit integrates a syringe that is propelled by a linear actuator. The volume of bioink expelled from each dispensing tip is manipulated volumetrically by this actuator. When bioink is introduced into the air-driven dispensing nozzle, pulsed air is injected, which propels the predetermined bioink volume out. The dispensing units work in coordination to manage both the dispensed droplets' locations on a substrate and the volume of each droplet. The bioprinter is additionally furnished with units to maintain temperature and humidity. In addition, the same volume of droplet can beMGH 2024-400-02 Quarles ref.125141.04818 dispensed via different drive pressure so that the interaction of the dispensed droplet with the existing bionic on the substrate can be controlled for different outcomes.

[0131] The open-source GRBL control system, encompassing GRBL firmware, Arduino Boards, and motor controllers, is utilized to oversee the bioprinter's multiple linear actuator motions. This 40-2 includes the XYZ gantry and the linear actuators that regulate the advancement of the syringe plungers. The activation of pulsed air is facilitated through the GRBL's built-in on-off control function. Standard GRBL-supported G-Codes and M-Codes are used to control the motion and the toggling on and off of the pulsed air. Codes are transmitted to the bioprinter via GRBL-compatible code senders (github.com / grbl / grbl / wiki / Using-Grbl). Lastly, a pressure regulator ensures the control of the pulsed air's pressure.

[0132] Cell culture and hydrogels

[0133] All animal procedures were approved by our Institutional Animal Care and Use Committee (IACUC). Whole articular stifle joints of immature calves were obtained from a local abattoir. The surface of each joint was disinfected with povidone-iodine, and the joint was opened using aseptic techniques. Articular cartilage tissues were collected, and superficial layers were removed. The harvested cartilage tissues were sliced into miniature pieces and digested overnight with 0.2% collagenase type II (Worthington) in DMEM-F12 medium (Invitrogen) at 37 °C. The digested tissues were filtered through cell strainers, and chondrocytes were collected and seeded onto 150 mm Petri dishes (Corning) in DMEM-F12 medium with 10% FBS (Corning) and 1% Penicillin / Streptomycin (Corning). When 80–90% confluence was reached, the chondrocytes were trypsinized and passaged for expansion. The same medium was used for all the following chondrocyte-related assays unless otherwise noted. Human mesenchymal stem cells (hMSCs) were obtained from the Stem Cell Repository at Texas A&M University and cultured in complete α-Minimum Essential Medium (αMEM, Gibco) supplemented with 15% fetal bovine serum (FBS, Atlanta Biologicals), 1% penicillin / streptomycin (Gibco) and 2 mM L-glutamine (Gibco) at 37 °C with 5% CO2. hMSCs were passaged when they reached 80% confluency. Medium was changed 2–3 times per week. 40-3 Both chondrocytes and hMSCs were harvested with 0.25% trypsin / EDTA and resuspended in a composite hydrogel containing collagen and fibrin before bioprinting.

[0134] Bioprinting ProcessMGH 2024-400-02 Quarles ref.125141.04818

[0135] General principle of cellular patterning using bioprinting

[0136] A bioink was made by mixing chondrocytes or hMSCs (6–10×106 cells ml-1 ) with collagen type I (2mg l-1 , Advanced BioMatrix) and fibrin (2mg ml-1 , Sigma) hydrogels. First, using a discrete dispenser, the bioink was dispensed to the surface of a Petri dish to form a droplet. Droplets containing thrombin (1–2 U ml-1 ) in DMEM-F12 medium without a hydrogel or cells were dispensed to the first droplet by higher pressure through a second dispenser. Higher-pressure- driven droplets without cells served as disrupting forces when they collided with the first droplet. These forces drove homogeneous cells into clusters while all the droplets merged into one larger droplet on the substrate. The thrombin quickly solidified the bioink, and the newly formed cell patterns were fixed in place without generating interfaces between clusters. This bioprinting process was repeated so that an array of micro-constructs was generated.

[0137] Optimize the bioprinter control parameters

[0138] The volumes of droplets, driving pressures, number of droplets, and droplet-dispensing locations are the four primary parameters controlling the cellular patterns within a micro-construct. Because there are material batch variations and fluctuations in the cell viscosity and concentration et al. among different assay batches, the same bioprinter control parameters can generate different patterns. Therefore, no universal parameters can be used to generate patterned micro-tissues with good repeatability among different batches of assays. However, within an assay, these 40-4 parameters are relatively stable. When the same bioprinter control parameters are used, good repeatability can be achieved within an assay.

[0139] The combinations of the different volumes of droplets, driving pressures, number of droplets and the location of droplet-dispensing are used as the bioprinter control parameters, and an array of micro-constructs can be bioprinted. The volume of droplets and the driving pressure are correlated with the sizes of cluster areas within a droplet. The number of droplets and the location of droplet-dispensing affect the uniformity of the clusters.

[0140] Therefore, a general protocol is to start a trial using a fixed droplet volume. The driving pressure gradually increases until apparent cellular clusters are observed after liquid colliding. This pressure value is set as low-end driving pressure. Then the driving pressure continues to be increased until the spherical shape of the cell-containing droplet is slightly disrupted, and 90% of this value is set as high-end driving pressure. Different levels of driving pressure can be selectedMGH 2024-400-02 Quarles ref.125141.04818 between the low and high-end pressure values. The volume of the droplet can also be varied. The number of droplets is usually chosen between efficiency and cluster uniformity‒use the least possible number of droplets to generate good uniformity. Each of the four parameters can be set to several different levels or values, and the parameters of their combinations are used to generate the array.

[0141] H-MCTs bioprinting

[0142] After the optimized PA-MCTs bioprinting parameters were chosen, the equivalent concentrations of cells and hydrogels in the PA-MCTs were calculated and were used to prepare cells containing hydrogels for H-MCTs bioprinting. H-MCTs used for comparing with corresponding PAMCTs were directly deposited onto the Petri dish in a two-droplets approach: cell-free thrombin solution in the first droplet and a cell-containing droplet in a composite hydrogel containing collagen 40-5 type I and fibrin. The lowest possible pressure was used for H-MCT bioprinting-calibrated for each assay batch. The final concentrations of cells and hydrogels were the same in corresponding PAMCTs and H-MCTs in each assay. The Petri dishes containing arrays of micro-constructs were transferred to an incubator to polymerize the collagen component fully.

[0143] Analysis of the distribution of the patterned clusters

[0144] Confocal imaging and chondrocyte pattern analysis: To analyze the size of the patterned cell clusters and the distance between the clusters, the same bioprinting protocol described above was used, except the diameter of each PA-MCT was controlled to be larger (2–3 mm) so that more cell clusters were analyzed. After the hydrogel component of the bioink was fully polymerized, samples were rinsed with PBS and stained with Calcein-AM and imaged under a confocal microscope (Eclipse Ti, Nikon). Acquired images were processed and analyzed using ImageJ. Equivalent circle diameters (ECDs) and maximal inscribed circles were calculated to represent the sizes of cell clusters and distances between clusters. Numbers of cell clusters of different sizes were counted, and the clusters were color labeled according to their sizes. To visualize cell patterns within a bioprinted PA-MCT, PA-MCTs were rinsed and stained with Calcein-AM (Invitrogen). The patterns of cells were imaged by combining fluorescence confocal microscopy and reflection confocal microscopy using a previously reported protocol.

[0145] Pattern analysis: We used the distributions of ECDs of cell clusters to represent the patterns of cell clusters within a bioprinted micro-construct. First, the ECD distributions were visualizedMGH 2024-400-02 Quarles ref.125141.04818 using Empirical 40-6 Cumulative Distribution Function (ECDF) plotting, which can also help select different clusters patterns. Kolmogorov-Smirnov distribution analysis was also used to choose unique patterns among the generated patterns.

[0146] Analyze the relationship between gene expression and the cellular cluster patterns: Replicates of each standard pattern were bioprinted using the same corresponding bioprinter control parameters. Only the replicates with the statistically same distribution as the corresponding standard distribution were included for further pattern analysis. Within the same assay, using the same lot of cells and hydrogels and the bioprinting parameters of the 20 unique patterns, 20 groups of PA-MCTs were bioprinted. The PA-MCTs were further cultured for 3weeks before qPCR analysis for gene expression of the cartilagenesis positively related Col-II, Sox-9 and ACAN and negatively related COL-I.

[0147] PCR scoring: The gene expression of the replicates of each gene in each group was averaged. Each gene expression was then normalized to the highest corresponding average value. The PCR score of an individual sample was calculated using the normalized values and the following equation: PCR score = PCR_COLII + PCR_Sox9 + PCR_ACAN - PCR_COLI

[0148] The PCR score of each pattern was the average score of all the replicates. All the 20 patterns were then ranked according to the averaged PCR scores in descending order. Each pattern was then named according to its ranking; for example, the group with the highest PCR score was named PAT1.

[0149] Area score: the summed area of clusters in a specific ECD range (Table S1) over the total cluster area, which reflects the percentage of cells under the influence of the patterns.

[0150] Number score: the summed number of clusters in a specific ECD range (Tale S1) over the total cluster numbers, reflecting patterning efficiency.

[0151] Pattern score = (Area score + Number score) / 2

[0152] Select the optimized ECD range using Spearman’s Rho correlation analysis: The pattern scores calculated using 66 different ECD ranges were ranked in descending order. Spearman's Rho rank correlation was performed between the pairs of PCR score ranking and each of the 66 pattern score rankings. The ECD range of the pattern score with the highest correlation with 40-8 the PCR score will be chosen as the optimized score range to select the optimized chondrogenesis pattern.MGH 2024-400-02 Quarles ref.125141.04818

[0153] Micro-tissue culture

[0154] After each micro-construct was fully solidified, medium was added to the Petri dishes. PAMCTs containing chondrocytes were cultured in DMEM / F12 (Gibco) supplemented with 10% FBS. PA-MCTs containing hMSCs were cultured in the following chondrogenic medium: high glucose (4.5 g l-1) Dulbecco's Modified Eagle's Medium (DMEM-HG, Invitrogen), 10% ITS + Premix Tissue Culture Supplement (Becton Dickinson), 10−7M dexamethasone (Sigma), 1 μM ascorbate-2-phosphate (Sigma), 1% sodium pyruvate (Invitrogen) and 10 ng ml-1 transforming growth factor-beta 1 (TGF-β1, Stemimmune LLC) up to 3 weeks. All the micro-constructs were cultured at 37 °C with 5% CO2.

[0155] Bio-assembly assays

[0156] Single PA-MCT migration assay: Individual PA-MCTs or H-MCTs were cultured for 2 weeks and then aspirated from the Petri dish and transferred to wells of a 96-well plate (one sample per well) and embedded with a mixed hydrogel composed of collagen and fibrin. After the fibrin component was quickly polymerized with thrombin, the 96-well plate was transferred to an incubator for further polymerizing the collagen component. After the hydrogels were fully solidified, 300 µl of culture medium was added to each well. All the samples were cultured at 37 °C with 5% CO2. After 24 h, the samples were washed with phosphate-buffered saline (PBS), fixed with paraformaldehyde, and stained with Alcian blue. At least triplicate samples were assayed.

[0157] PA-MCTs and H-MCTs bio-assembly assay with less than 20 MCTs: PA-MCTs or H-MCTs were cultured for 2 weeks and then collected and transferred into the wells (< 20 MCTs / well) of a round-bottom 96-well plate so that they attached to each other within a well and covered with a composite hydrogel composed of collagen and fibrin. After the hydrogel was quickly polymerized with thrombin, the plate was transferred to an incubator for further polymerizing the collagen component. After the hydrogel in each well was fully solidified, each sample was transferred to a well of a 24 well plate. Two ml of culture medium was added to each well, and the samples were cultured at 37 °C with 5% CO2 for 5 weeks. The assembly dynamic was tracked daily under an inverted microscope (Nikon), and at least triplicate samples were terminated each week for histological analysis.

[0158] PA-MCTs and H-MCTs bio-assemble into large macro-cartilage: PA-MCTs and H-MCTs were collected after 2 weeks of culture. PA-MCTs or H-MCTs were added to transwellsMGH 2024-400-02 Quarles ref.125141.04818 (~500 / well) and covered with a mixed hydrogel composed of collagen and fibrin. The hydrogel was quickly polymerized with thrombin and transferred to an incubator for further polymerization. After the hydrogel was fully solidified, the medium was added to the well to cover both under and above the constructs. Two ml of culture medium was added to the well and the samples were cultured at 37 °C with 5% CO2 for 5 extra weeks. The medium was changed twice per week. At least triplicate samples were assayed for each condition and each group.

[0159] Bio-assembly tracking using live imaging: PA-MCTs bioprinted using hMSCs were placed in a glass-bottom Petri dish and covered with a mixed hydrogel composed of collagen and fibrin. The hydrogel was quickly polymerized with thrombin and transferred to an incubator for further polymerization. After the hydrogel was fully solidified, medium was added to the well. The dynamic of the bio-assembly process of PA-MCTs was tracked and recorded using an inverted microscope at 37 °C with 5% CO2.

[0160] Integration assays

[0161] Harvesting and decellularizing articular cartilage, bone, and osteochondral tissues: Cartilage, bone and osteochondral tissues were removed from bovine diarthrodial joints and processed into cylindrical and cuboid tissues using stainless steel punches and an oscillating saw. The cuboid bone tissues were flushed with pressured water to remove bone marrow and other debris. The tissues were placed in a jar filled with a 1% sodium dodecyl sulfate solution and were processed on an orbital shaker to remove cells. Sheep stifle joints were obtained locally and also from sacrificed sheep in other research studies as approved by our IACUC. The stifle joints were used for modeling PA-MCT delivery and repairing large cartilage defects. For the latter, intact individual condyles were removed from stifle joints and decellularized using the same protocol described above. The intact distal ends of the femurs of mature rabbits were removed using an oscillating saw, and all tissues superficial to the bone were removed with a scalpel and decellularized using the same protocol described above. After the decellularization process, all the tissues were rinsed with distilled water and PBS sequentially and were sterilized with 70% ethanol before use.

[0162] Integration assay on cylindrical cartilage and bone tissues: An agarose solution was cast into wells of 12-well plates and solidified. Holes were punched in the agarose gels to match the sizes of cylindrical cartilage or bone tissues generated previously. PA-MCTs and H-MCTs wereMGH 2024-400-02 Quarles ref.125141.04818 cultured for 2 weeks and then collected from the Petri dish. PAMCTs or H-MCTs were placed on top of the cylindrical tissues and covered with a mixed hydrogel composed of collagen and fibrin. The hydrogel was quickly polymerized with thrombin, and the plates were transferred to an incubator for further polymerizing the collagen component. After the hydrogel was fully solidified, medium was added to the wells and samples were cultured at 37 °C with 5% CO2. After 1–2 days of culture, constructs including the bone or cartilage tissues and the added PA-MCTs or H-MCTs were removed from the agarose wells and transferred to wells of 6-well plates and cultured for 5 extra weeks. The medium was changed twice a week, and at least triplicate samples were assayed for each condition and group.

[0163] Repairing cartilage defects in an osteochondral explant model

[0164] Surface contour fitting assay: As shown in FIGS. 18A-18B (Supporting Information), silicone sheets with convex or concave edge surfaces were placed between two glass slides and held vertically. PA-MCTs were gently placed over the surfaces. To evaluate if the PA-MCTs could self-fit to the curvature of the surfaces, images were taken using a stereo-microscope which was perpendicularly orientated to the glass slides and evaluated by two independent scientists.

[0165] Medium size defect repair:

[0166] As shown in FIG.21, cuboid decellularized osteochondral explants were used in this assay. A full-thickness defect 5 mm in diameter and 3 mm in depth (down to the subchondral bone) was created in each decellularized osteochondral explant using a drill (the red dotted region). A partial- thickness defect was also created from the edge of the full-thickness defect to the edge of the explant using a scalpel (the green dotted region). The explants were rinsed with sterile saline to remove any debris. Approximately 300 PA-MCTs or H-MCTs per explant were added and covered with a mixed hydrogel composed of collagen and fibrin. The hydrogel was quickly polymerized with thrombin, and each explant was transferred to an incubator for further polymerization. After the hydrogel was fully solidified, medium was added and each explant was cultured at 37 °C with 5% CO2 for 5 extra weeks. The medium was changed twice a week and at least triplicate samples were assayed for each group.

[0167] Large defect repairMGH 2024-400-02 Quarles ref.125141.04818

[0168] Analysis of the injectability of PA-MCTs: PA-MCTs of various sizes in a mixed hydrogel composed of collagen and fibrin were manually injected through glass tubes of various IDs using a syringe. The smallest ID of the glass tube tested was 1.4 mm, and the largest diameter of the PA- MCTs tested was ~650 µm. The injection process was monitored under an inverted microscope (Nikon). The injection of PAMCTs to a cartilage defect in a sheep stifle joint was performed to mimic surgical implantation.

[0169] Injecting PA-MCTs to the defects: Irregularly-shaped (water-drop-like shape) large defects were created in decellularized sheep condyles using a curette. Each defect was made at least 11 mm in the long axis and down to the subchondral bone. Approximately 800 PA-MCTs were injected into each defect in a mixed hydrogel composed of collagen and fibrin. The injection device (~3.3 mm OD and ~2.5 mm ID, FIG.16) was made by slicing off the proximal section of a repeat pipette tip (Eppendorf). After injection, the hydrogel was quickly polymerized with thrombin, and then each condyle was transferred to an incubator for further polymerization. After the hydrogel was fully solidified, medium was added, and samples were cultured for extra 5 weeks. At least triplicate samples were assayed.

[0170] Generating customized osteochondral allografts

[0171] De-identified computed tomography images of a human knee, which were stock in our lab, were imported to 3D Slicer software, and the 3D volumetric reconstruction was performed and exported as an STL file. The STL file was further processed using Blender software: (i) generating a cartilage layer from the bone surface, (ii) generating a cartilage defect in the weightbearing region of the medial femoral condyle, (iii) mimicking the preparation process of the defect site, and (iv) designing an osteochondral allograft 1 cm in diameter. Decellularized bone plugs (curved top, 1 cm in diameter and 8 mm in height) were created using the same protocol as described above, matching the design of the plugs in the 3D model. Following the same protocol as described above: (i) bone plugs were placed into agarose wells; (ii) PA-MCTs were cultured for 2 weeks and then collected and placed onto the bone plugs together with a mixed hydrogel composed of collagen and fibrin; and (iii) the constructs were further cultured in the medium for 5 extra weeks and the fidelity was evaluated by two independent scientists.

[0172] Resurfacing an entire condyle by PA-MCT bio-assemblyMGH 2024-400-02 Quarles ref.125141.04818

[0173] Femoral condyles were removed from the hind limbs of adult New Zealand White rabbits that were humanely euthanized. Cartilage and other soft tissues were removed from the condyles with a scalpel. The condyles then went through the decellularization process mentioned above and were sterilized with 70% ethanol and rinsed with PBS. To resurface a condyle, two layers of ~800 PA-MCTs after 2 weeks of culture, were manually applied to the bony surface together with a small volume of a mixed hydrogel composed of collagen and fibrin. The PA-MCTs attached to the bony surface without extra support. The constructs were cultured in the medium at 37 °C with 5% for 5 extra weeks.

[0174] Drug screening

[0175] Femoral condyles were removed from the hind limbs of adult New Zealand White rabbits that were humanely euthanized. Cartilage and other soft tissues were removed from the condyles with a scalpel. The condyles then went through the decellularization process mentioned above and were sterilized with 70% ethanol and rinsed with PBS. To resurface a condyle, two layers of ~800 PA-MCTs after 2 weeks of culture, were manually applied to the bony surface together with a small volume of a mixed hydrogel composed of collagen and fibrin. The PA-MCTs attached to the bony surface without extra support. The constructs were cultured in the medium at 37 °C with 5% for 5 extra weeks.

[0176] The ranking of cartilage positively related genes was based on the sum of gene expressions of Sox9, collagen type II and ACAN, where the rank score of the highest value is 4 and the lowest value is 1.

[0177] The ranking of inflammatory genes was based on the sum of gene expressions of MMP-3 and MMP-13, where the rank score of the lowest value is 4 and the highest value is 1. The 40-15 ranking of overall anti-inflammatory effects was evaluated by combining the ranking score over GAG contents, expression of inflammatory genes and cartilage positively related genes.

[0178] Microinjection

[0179] Grooves were generated in an agarose gel using a mold. PA-MCTs were aspirated and transferred into the grooves, and individual PA-MCTs were manually adjusted to be spread apart from each other. A pre-pulled glass pipette (WPI) was mounted on a micromanipulator and connected to a syringe pump. A solution of 10 µl of FITC-dextran was aspirated into the glassMGH 2024-400-02 Quarles ref.125141.04818 pipette. Under a stereo-microscope and using a micromanipulator, the tip of the glass pipette was inserted into one PA-MCT at a time, and the FITC-dextran was injected. Because a PA-MCT contains dense matrix and the pressure inside it is high, the volume of the injected solution was controlled by the pump execution time. The size of the yellow circle (bright field color of FITCdextran solution) was also used as a secondary volume indicator. The success of injecting FITCdextran was determined by fluorescence microscopy.

[0180] Cell viability assay

[0181] A calcein-AM (Invitrogen) staining assay was used to evaluate cell viability within the first 3 days post bioprinting. A CellTiter-Glo 3D Cell Viability (Promega) assay was used to analyze cell Ranking Score IL-1β+DEX IL-1β+IGF-1 IL-1β+TGFβ IL-1β+CEL Cartilage-Positive-Genes 23 41 Anti Inflammation-Genes 4321 GAG Content 4321 Overall 1098340-16 viability from the 4th day post bioprinting, and data were normalized to DNA content, which was measured by a PicoGreen assay (Promega). All the assays were performed according to instructions from the manufacturers.

[0182] Macroscopic Imaging

[0183] Macroscopic images were taken under a stereo-microscope equipped with a 5MP digital camera (Motic). The magnification scale was calculated each time with a calibration slide placed at the same focal depth as the sample.

[0184] Histological Staining

[0185] Tissue harvest and process: Samples without bone tissues, namely individual PA-MCTs, H-MCTs and bio-assembled macro-tissues, were collected, rinsed with PBS, fixed with paraformaldehyde, dehydrated through a graded ethanol series, embedded in paraffin, and sectioned into slices of 5 µm thickness using a microtome (Microm). After fixation, all other samples consisting of bone tissues were decalcified with formic acid and then were processed through the same procedures as above.

[0186] Safranin O and Alcian blue staining

[0187] Slides were deparaffinized with Xylene Substitute (Leica) and were rehydrated through a graded ethanol series. An Alcian blue solution (counterstained with fast red, Sigma-Aldrich) and a Safranin O solution (counterstained with fast green, Sigma-Aldrich) were used to stain sulfatedMGH 2024-400-02 Quarles ref.125141.04818 glycosaminoglycans (sGAG). Both Alcian blue and Safranin O staining were performed for most of the samples.

[0188] GAG semi-quantification by image processing

[0189] Color intensities in a blue or red channel of slides stained with Alcian blue or Safranin O solutions have been reported to correlate with GAG content. To determine linearity and the degree of correlation between dye concentrations (Alcian blue and Safranin O) and color intensities, stock dye solutions were diluted into a series of concentrations and pipetted into a 96- well plate. The solutions were imaged under a microscope through a 5-megapixel color camera. The identical condition was utilized for each image acquisition, including the illuminating light intensity (a LED light source was used to precisely control light illumination), objectives, exposure time, and white balance. Relative GAG content was quantified by calculating color intensities within the blue (Alcian blue) or red (Safranin O) channel of captured images respectively, using ImageJ. With the coefficient of determination (R2) being 0.9961, we found that the dye concentration highly correlates with the color intensity.

[0190] To analyze relative GAG content in tissue sections, slides were stained with Alcian blue and Safranin O solutions, imaged and analyzed using the identical protocol described above. Specifically, to study the mechanism of PA-MCT bio-assembly and to compare the GAG content inside and between PA-MCTs, pixel intensities along a series of parallel lines spanning inside and outside spaces of the PA-MCTs were analyzed (FIGS. 11A-11B). To evaluate the quantity of neocartilage inside articular cartilage defects and to analyze the distribution of GAG content throughout neocartilage inside the defects, 2D pixel intensity mapping was performed using ImageJ.

[0191] Immunohistochemistry

[0192] Slides were incubated in a citrate buffer solution in a microwave to retrieve antigens. Nonspecific binding was reduced by pre-incubating slides with a bovine serum albumin solution. 40-18 Slides were then incubated with an anti-collagen type II antibody (CIIC1, Developmental Studies Hybridoma Bank) at 4 °C overnight. They were further incubated with an Alexa Fluor 488- labeled anti-mouse secondary antibody (Bio legend) in 5% (w / v) bovine serum albumin (BSA) for 12 h at room temperature. Slides were then mounted and imaged under a fluorescence microscope (Nikon) equipped with a monochrome camera (SPOT Imaging). Similar to the semiquantificationMGH 2024-400-02 Quarles ref.125141.04818 of GAG content, the content of collagen type II was determined by analyzing the color intensities of the fluorescent images using ImageJ.

[0193] Whole-mount Alcian blue staining

[0194] Assembled macro-tissues were rinsed with PBS, fixed with paraformaldehyde and rinsed with PBS again. Each sample was then submerged in an Alcian blue solution for GAG staining and rinsed extensively to remove any unbound dye.

[0195] Scoring bioprinted individual PA-MCTs and H-MCTs and defect repair

[0196] Table 3. Modified Bern Score system for evaluating individual bioprinted PA-MCTs and H-MCTs (minimum score: 0; maximum score: 9; section: 5 ^m thickness)MGH 2024-400-02 Quarles ref.125141.04818

[0197] Table 4. Modified O'Driscoll Score for evaluating osteochondral defects repair using PA- MCTs and H-MCTs[8] (Minimum Score: 0; Maximum Score: 22; Section 5 µm thick)MGH 2024-400-02 Quarles ref.125141.04818MGH 2024-400-02 Quarles ref.125141.04818

[0198] DMMB assay

[0199] A dimethyl methylene blue assay (DMMB) was used to determine the content of GAG in PA-MCTs and H-MCTs. In brief, PA-MCTs, H-MCTs, assembled macro-tissues and native articular cartilage were collected, rinsed with PBS and digested with papain. The digested solution was reacted with DMMB (Sigma-Aldrich), and absorbance was measured at 492 nm. Using the absorbance of standard chondroitin sulfate (Sigma-Aldrich) as a standard curve, GAG content in the samples was calculated accordingly.

[0200] To compare the homogeneity between native articular cartilage explants and PA-MCTs, explants—at a typical size of 3 mm in diameter and 1 mm in height—were harvested from the medial femoral condyle, lateral femoral condyle, medial tibial plateau and lateral tibial plateau (triplicate samples were harvested from each location). Relative GAG content in the explants and PA-MCTs was analyzed using the DMMB assay described above. The coefficient of variation of the GAG content of PA-MCTs was calculated using DMMB-readouts of at least 5 randomly selected samples. The coefficient of variations of the GAG content of native cartilage explants was calculated using DMMB-readouts of samples from each region or the entire knee.

[0201] Quantitative real-time PCR

[0202] To examine the specific gene expression listed in Table 5, samples were collected and rinsed with PBS. Total RNAs in PA-MCTs and H-MCTs were extracted using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. Samples were treated with DNase I removing any possible genomic DNA contamination. Concentrations and purities of the RNA samples were determined by a NanoDrop 2000 spectrophotometer (Thermo scientific). Total RNAs were reverse-transcribed into cDNAs using a SuperScript III kit (Invitrogen) following the 40-22 manufacturer's instruction. Real-time PCR was performed using a SYBR Green PCR Master Mix (Applied Biosystems) in a thermal cycler. Gene expression of collagen type II (COL2A1), collagen type I (COL1A1), aggrecan (ACAN), Sox 9, MMP-3 and MMP-13 was analyzed. The primer sequences are listed in Table 5. Articular cartilage tissues from the same bovine source were usedMGH 2024-400-02 Quarles ref.125141.04818 as a positive control in the PCR reaction. mRNA expression levels of the target genes were normalized to that of the 18S rRNA reference gene. Relative expression of the genes of interest was quantified using a ΔΔCT method.

[0203] Table 5. Forward and reverse primers used qRT-PCR analysis Species Gene Name Primer Sequence (5'–3') SEQ ID NO:

[0204] Mechanical TestsMGH 2024-400-02 Quarles ref.125141.04818

[0205] Micropipette Aspiration: A micropipette aspiration technique was used to measure the stiffness of individual PAMCTs or H-MCTs using a protocol reported previously with some modification. In short, as shown in FIG. 22 below, 2 / 3 of a glass micropipette was mounted horizontally inside a Petri dish placed on an inverted microscope stage (Nikon), while the remaining 1 / 3 was extruded outside the Petri dish via a drilled hole through the wall of the dish. The end inside the Petri dish was open, and the other end was connected to a negative pressure source through Tygon tubing. Pressure differences between the two ends of the micropipette (ΔP) were recorded via a digital pressure meter. To measure the hardness of PA-MCTs or H-MCTs, one PA-MCT or H-MCT at a time was placed at the open end of the micropipette under a workstation magnifier. Monitored from the inverted microscope, the PA-MCT or H-MCT was aspirated and quickly sealed the open end of the glass micropipette when an initial ~1 kPa negative pressure was applied. The pressure 40-24 was then changed at a constant speed of 2 kPa / step, and an image was captured per step of pressure change. The inner radius of the glass micropipette was denoted as Ri; the length of the tongue inside the pipette was denoted as X, and the initial X was denoted as X0. The current assay was terminated when -90 kPa pressure was reached or when X > Ri. Pressure differential (ΔP) versus normalized deformation (X – X0) / Ri was plotted, and stiffness values of PA-MCTs and H-MCTs were calculated by curve fitting.

[0206] Confined and unconfined compression tests: In a confined compression test (FIGS.23C- 23D), specimens were placed in a PBS bathed testing chamber inside a universal testing machine which is comprised of a ball-screw guide rail driven by a servo motor (Panasonic) and a displacement encoder with the resolution of 0.5 µm (Panasonic) and load cells (HBM). The chamber base is made of impermeable stainless steel with a center circular well. Samples were punched into disks to match the inner diameter of the well for a tight contact when placed into the well. The cartilage surface was compressed with a stainless-steel porous indenter at a speed of 1 µm s-1 until a 20% strain was reached. The sample was allowed for stress relaxation to equilibrium that was determined when the change of stress 40-25 was less than 0.003 MPa over 180 s or when the relaxation was over 3600 s. Aggregate modulus HA and Permeability k0 were then calculated using a biphasic model.

[0207] An unconfined compression test (FIGS.23A-23B) was used to determine Young's Modulus using the identical set-up to the confined compression test except specimens were compressed between two non-permeable platens.MGH 2024-400-02 Quarles ref.125141.04818

[0208] Push-out test

[0209] As shown in FIGS. 24A-24D, upon completing a defect repairing assay, cartilage proportions were sliced off the constructs (FIG. 24A), and the height of each removed cartilage was measured (FIG.24B). The removed cartilage tissues were, one tissue at a time, placed into a specimen holder (FIG.24C) and transferred to the universal testing machine (FIG.24D) that was described above. To test the adhesion force between each neocartilage and the surrounding native cartilage, a rigid 4 mm diameter indenter was moved down to push the center of the specimen at 1µm s-1 until a tissue separation occurred. The maximum force was normalized by the lateral area of the interface between the neocartilage and native cartilage. Each resulting value was recorded as the failure stress.

[0210] Statistical analysis

[0211] Data were statistically analyzed using JASP (University of Amsterdam). As appropriate, one-way ANOVA in conjunction with Tukey post hoc analysis was used to determine differences among groups, and the independent-samples t test was used to determine differences between two groups. Levene's test was used to determine differences of the coefficient of variations among groups. A P-value below 0.05 was considered to be significantly different.

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Claims

MGH 2024-400-02 Quarles ref.125141.04818 CLAIMS What is claimed is:

1. An encapsulated microsphere comprising a plurality of cells in a matrix, wherein the plurality of cells is arranged in clusters of cells within the encapsulated microsphere.

2. The encapsulated microsphere of claim 1, wherein the clusters of cells are dispersed within the encapsulated microsphere without an interface separating the clusters of cells.

3. The encapsulated microsphere of claim 1, wherein the matrix comprises a fibrin matrix.

4. The encapsulated microsphere of claim 1, wherein the plurality of cells comprises chondrocytes, mesenchymal stem cells, or a combination of chondrocytes and mesenchymal stem cells.

5. The encapsulated microsphere of claim 1, wherein the encapsulated microsphere comprises 2 to about 1x106chondrocytes.

6. The encapsulated microsphere of claim 1, wherein the encapsulated microsphere has a diameter of about 1 ^m to about 1 mm.

7. The encapsulated microsphere of claim 1, wherein the encapsulated microsphere has a diameter of about 100 ^m to about 700 ^m.

8. The encapsulated microsphere of claim 7, wherein the encapsulated microsphere has a diameter of greater than about 400 ^m to less than about 650 ^m.

9. The encapsulated microsphere of claim 1, wherein the clusters of cells have an average equivalent circular diameter of about 20 ^m to about 200 ^m.

10. The encapsulated microsphere of claim 1, wherein the cells in the encapsulated microsphere further comprise an exogenous polynucleotide.

11. The encapsulated microsphere of claim 10, wherein the exogenous polynucleotide comprises a sequence encoding a polypeptide.

12. The encapsulated microsphere of claim 11, wherein the polypeptide comprises a nucleic acid-guided nuclease.MGH 2024-400-02 Quarles ref.125141.04818 13. The encapsulated microsphere of claim 12, wherein the nucleic acid-guided nuclease comprises a Cas nuclease.

14. The encapsulated microsphere of claim 13, wherein the Cas nuclease comprises Cas9.

15. The encapsulated microsphere of claim 12, wherein the encapsulated microsphere further comprises a sequence encoding a guide RNA (gRNA).

16. A tissue construct comprising a plurality of the encapsulated microspheres of claim 1.

17. The tissue construct of claim 16, wherein the plurality of encapsulated microspheres is 3 to 1000 encapsulated microspheres.

18. The tissue construct of claim 17, wherein the tissue construct is about 1 mm to about 10 mm in diameter.

19. The tissue construct of claim 18, wherein the tissue construct is about 9 mm in diameter.

20. The tissue construct of claim 16, wherein the tissue construct comprises smooth white surfaces and / or glycosaminoglycan deposition throughout the construct, optionally, as detected by alcian blue staining.

21. An orthopedic graft comprising a plurality of the encapsulated microspheres of claim 1 affixed to cartilage, a bone, or a bone fragment.

22. The orthopedic graft of claim 21, wherein the plurality of encapsulated microspheres are affixed to the bone or the bone fragment in a matrix comprising fibrin.

23. The orthopedic graft of claim 22, wherein the matrix further comprises thrombin or collagen type I.

24. The orthopedic graft of claim 21, wherein the bone or bone fragment is decellularized.

25. A method of generating encapsulated microspheres, the method comprising, at an apparatus comprising a first dispensing unit comprising a first nozzle and a second dispensing unit comprising a second nozzle, (a) forming a first droplet at the first nozzle comprising at least one cell and a first matrix and forming a second droplet at the second nozzle comprising a second matrix;MGH 2024-400-02 Quarles ref.125141.04818 (b) dispensing the first droplet onto a substrate and dispensing the second droplet onto the substrate such that the first droplet and the second droplet collide to form an encapsulated microsphere, wherein the second droplet is dispensed at a velocity sufficient to disrupt the arrangement of cells in the first droplet, thereby generating clusters of cells within the encapsulated microsphere, and wherein the first matrix and the second matrix comprise reagents that form a gel or a solid following the collision of the first droplet and the second droplet.

26. The method of claim 25, wherein the first matrix comprises fibrin and the second matrix comprises thrombin or the first matrix comprises thrombin and the second matrix comprises fibrin.

27. The method of claim 25, wherein the first matrix comprises fibrin and the second matrix comprises thrombin.

28. The method of claim 25, wherein the first droplet is dispensed such that the cells achieve a relatively homogenous distribution in the droplet.

29. The method of claim 25, wherein there is no interface between the clusters of cells within the encapsulated microsphere.

30. The method of claim 25, wherein at least one cell comprises a chondrocyte.

31. The method of claim 25, wherein the concentration of the cells in the first droplet is about 6x106cells / ml to about 10x106cells / ml.

32. The method of claim 25, wherein the concentration of the fibrin is about 2 mg / ml.

33. The method of claim 25, wherein the concentration of thrombin is about 1 unit / ml to about 2 units / ml.

34. The method of claim 25, wherein either the first of the second matrix comprises collagen type I.

35. The method of claim 34, wherein the concentration of collagen type I is about 2 mg / l.

36. The method of claim 25, wherein the first and second dispensing unit each comprise a syringe includingMGH 2024-400-02 Quarles ref.125141.04818 (i) a hollow body and a plunger dimensioned to translate in the body, the body having an exit orifice; (ii) an actuator in contact with a proximal end of the plunger; (iii) a controller for controlling linear motion of the actuator wherein the controller executes a program received from a computer in the controller to drive the actuator toward the proximal end of the plunger to dispense a bioink from the body of the syringe; and (iv) a nozzle having a wall defining a fluid path extending from an inlet of the nozzle to an outlet of the nozzle, the inlet of the nozzle being in fluid communication with the exit orifice of the body of the syringe, wherein as the controller drives the actuator toward the proximal end of the plunger bioink flows into the fluid path, wherein the nozzle includes a fluid passageway in fluid communication with a source of fluid and the fluid path, wherein the source of fluid comprises a controllable valve for supplying pulsed fluid from the source of fluid to the fluid passageway to create separate droplets from the flow of bioink in the fluid path in the nozzle by blowing the droplets away from the nozzle using the pulsed fluid from the source of fluid.

37. The method of claim 25, wherein the dispensing comprises blowing the droplets away from the nozzle using pulsed fluid from the source of fluid.

38. The method of claim 37, wherein the pulsed fluid comprises a gas.

39. The method of claim 38, wherein the pulsed fluid comprises pulsed air.

40. The method of claim 39, wherein the air comprises millisecond pulsed air.

41. The method of claim 25, wherein a volume of each droplet is in a range of about 10 nanoliters to about 15 microliters.

42. The method of claim 25, wherein the method further comprises repeating steps (a) and (b) n additional times, wherein n is an integer greater than 1, to generate n encapsulated microspheres.

43. The method of claim 25, wherein the first droplet comprises 2 to about 1x106chondrocytes.MGH 2024-400-02 Quarles ref.125141.04818 44. The method of claim 25, wherein the encapsulated microspheres have a diameter of about 1 ^m to about 1 mm.

45. The method of claim 44, wherein the encapsulated microspheres have a diameter of about 100 ^m to about 700 ^m.

46. The method of claim 45, wherein the encapsulated microspheres have a diameter of greater than about 400 ^m to less than about 650 ^m.

47. The method of claim 42, wherein the n encapsulated microspheres are arranged on the substrate to generate a tissue construct precursor.

48. The method of any one of claims 25-46, wherein the substrate is a partial-thickness defect in articular cartilage.

49. The method of any one of claims 25-46, wherein the substrate is a full-thickness defect in articular cartilage.

50. The method of claim 47, wherein the tissue construct precursor is further cultured in vitro for about 1 day to about 10 weeks to generate a tissue construct.

51. The method of claim 42, wherein the method further comprises applying the n encapsulated microspheres to a cartilage defect.

52. The method of claim 51, wherein applying comprises depositing the n encapsulated microspheres in a delivery matrix.

53. The method of claim 52, wherein the delivery matrix comprises fibrin.

54. The method of claim 53, wherein the delivery matrix further comprises collagen type I.

55. The method of any one of claims 52-54, wherein the method further comprises applying a solidifying matrix comprising thrombin to the delivery matrix to generate a solidified matrix comprising the encapsulated microspheres.

56. The method of any one of claims 25-55, wherein the method further comprises repeating steps (a) and (b) n times and varying at least one parameter of encapsulated microsphere formation in each of the n repeats, to generate n encapsulated microspheres with varying properties;MGH 2024-400-02 Quarles ref.125141.04818 analyzing at least one parameter related to cartilagenesis in each of the n encapsulated microspheres; and selecting at least one parameter of encapsulated microsphere formation, based on the analysis of the at least one parameter related to cartilagenesis.

57. The method of claim 56, wherein the at least one parameter of encapsulated microsphere formation comprises pulsed fluid pressure of the first and or second nozzle.

58. The method of claim 56, wherein the at least one parameter related to cartilagenesis comprises equivalent circular diameter of the clusters in the encapsulated droplet or expression of at least one gene or protein.

59. The method of claim 58, wherein the at least one gene or protein comprises collagen type II (COL-II), transcription factor SOX-9 (SOX9), aggrecan (ACAN), or collagen type I (COL-I).

60. The method of claim 59, wherein at COL-II, SOX9, and ACAN are positively related to cartilagenesis and COL-I is negatively related to cartilagenesis.

61. A Method of testing a candidate therapeutic, the method comprising contacting the candidate therapeutic to at least one of the encapsulated microspheres of claim 1.

62. The method of claim 61, wherein the method further comprises detecting at least one parameter associated with performance of the candidate therapeutic.

63. The method of claim 62, wherein detecting the at least one parameter comprises detecting gene expression, protein expression, glycosaminoglycan (GAG) deposition, or protein secretion.

64. The method of claim 61, wherein the method further comprises contacting the at least one encapsulated microsphere with an inflammatory reagent.

65. The method of claim 64, wherein the inflammatory reagent comprises interleukin 1 beta (IL-1^).

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