Cellular graft delivery system, method for treating diseases involving collagen XII expression and cell graft composition - Patent Application 20070122997

Abstract

A cell graft delivery system capable of delivering a cell graft to a target tissue is provided. [Solution] The present invention relates to a cell graft delivery system for delivering cell grafts to target tissues, comprising a cell graft bound to a novel BiFP (bifunctional peptide) with tissue targeting and cell differentiation instruction functions. The BiFP (bifunctional peptide) of the present invention is constructed from a peptide containing a collagen XII targeting sequence that mediates the tissue targeting function and an integrin / DDR binding motif sequence that mediates the cell binding and cell differentiation instruction functions.

Description

[Technical Field]

[0001] The present invention relates to a cell transplant and its delivery system for regenerative medicine. More specifically, the present invention relates to the use of stem cell transplants delivered in a delivery system constructed with bivalent peptides, including collagen-like peptides (CLPs), which have tissue targeting and cell fate determination functions. [Background technology]

[0002] Collagen, the most abundant extracellular matrix (ECM) protein in the body, is widely used in biomedical research and clinical practice. However, animal-derived collagen is limited by its high immunogenicity and pathogenicity (Lynn, A.K., et al. J Biomed Mater Res B Appl Biomater 71, 343-354, 2004). Instead, the development of synthetic, performance- and structurally optimized collagen-like materials derived from supramolecular assemblies of building blocks, such as collagen-like peptides (CLPs), has been applied to 3D cell culture and tissue engineering (Prince, E. & Kumacheva, E. Nature Reviews Materials 4, 99-115, 2019). The fibrous structure of these materials mimics the filament structure of the ECM and determines their biomechanical properties, signaling functions, and cellular command cues (Chau, M., et al. Advances in Polymer Science 268, 167-208, 2015). In addition to fiber morphology and biophysical properties, the organization of adhesive ligands may also influence how cells interact with these synthetic materials, potentially facilitating regenerative medicine (Boekhoven, J. & Stupp, SI Adv Mater 26, 1642-1659, 2014). Another contribution that synthetic collagen-like materials should offer to the success of cell therapy and regenerative medicine is the homing of transplanted cells to target tissues, such as those used in controlled drug delivery or genome editing technologies (Li, J. & Mooney, DJ Nature Reviews Materials 1, 16071, 2016; Tong, S., et al. Nature Reviews Materials 4, 726-737, 2019). However, this remains an unmet need in this field.

[0003] In this study, we developed a bifunctional peptide (BiFP) for regenerative medicine of osteoarthritis (OA) and corneal epithelial defects. This CLP possesses both OA cartilage and corneal stroma targeting and cell fate control functions. The OA cartilage and corneal stroma targeting function is mediated by a specific peptide sequence that targets collagen XII, an extracellular matrix protein specifically expressed in OA cartilage and corneal stroma. Meanwhile, the cell differentiation instruction function is mediated by integrin or DDR binding motifs, which have previously been shown to induce chondrogenic differentiation of human MSCs.

[0004] Our previous patent application, TW202128730, demonstrated that a collagen XII targeting peptide (Col12-TP) can deliver MSCs to damaged articular cartilage when conjugated with hyaluronic acid (HA). However, this approach requires that HA be methacrylated with approximately 28% uptake rate and then conjugated with Col12-TP via Michael addition chemistry. MSCs express CD44 on their surface to bind to HA. This strategy requires high chemical conjugation efficiency and is dependent on CD44 expression. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the present invention attempts to design bifunctional peptides (BiFPs) containing a triplicate of CLP sequences and an integrin or DDR-binding motif flanked by Col12-TP sequences to deliver cell grafts to target tissues when bound to the grafts.

[0006] Based on the above objectives, the present invention demonstrates that engineered BiFP can bind to MSCs and deliver them to target tissues, such as the surface of OA cartilage and the corneal stroma, for OA and corneal epithelial regeneration. Furthermore, BiFP binding dose-dependently promotes MSC viability, proliferation, and chondrogenic differentiation. [Means for solving the problem]

[0007] Thus, one aspect of the present invention relates to a cell graft, or cell graft composition, delivered in a delivery system. The composition includes a cell graft bound to a BiFP (bifunctional peptide) for delivery of the cell graft to a target tissue. BiFP consists of a tissue targeting sequence and a cell binding motif sequence flanked by GPO repeats. In some embodiments, the target tissue expresses collagen XII, including osteoarthritic (OA) or degenerative intervertebral discs, corneal epithelium, infarcted cardiac tissue, skin dermis, and peri-hair follicle tissue.

[0008] In some embodiments, the tissue-targeting sequence is an osteoarthritis (OA) or degenerative disc-targeting sequence. In some embodiments, the cell binding sequence is an integrin binding motif or a DDR binding motif.

[0009] In some embodiments of the invention, the BiFP comprises a collagen XII targeting sequence, an integrin α2β1-binding motif sequence or a DDR-binding motif sequence, and at least three copies of GPO flanked by the integrin-binding motif or the DDR-binding motif.

[0010] In some embodiments of the present invention, the integrin motif is an α2β1 integrin binding motif. In one embodiment, the α2β1 integrin binding motif has the sequence GFOGER.

[0011] In another embodiment of the invention, the cell binding motif is a DDR binding motif. In one embodiment, the DDR binding motif has the sequence GVMGFO.

[0012] In one embodiment, the BiFP has the sequence GPOGPOGPOGPOGFOGERGPOGPOGPOGPODLQYWYPIWDTH. In another embodiment, the BiFP has the sequence GPOGPOGPOGPOGVMGFOGPOGPOGPOGPODLQYWYPIWDTH.

[0013] In some embodiments of the present invention, the cell grafts include, but are not limited to, MSCs, musculoskeletal progenitor or differentiated cells, and corneal cells or their progenitor cells. In one embodiment, the cell graft is an autologous MSC graft. In other embodiments, the cell graft is an allogeneic MSC graft.

[0014] Another aspect of the present invention relates to a method for treating a disease in which expression of collagen XII is implicated. The method comprises administering to a subject in need thereof a therapeutically effective amount of a cell graft delivered together with BiFP (a bifunctional peptide), where BiFP consists of a tissue targeting sequence and a cell binding motif sequence flanked by GPO repeats. In some embodiments of the present invention, diseases involving collagen XII expression include, but are not limited to, musculoskeletal disorders, sepsis or sterile keratitis, dry eye syndrome, heart disease, skin defect wounds, and diseases involving hair follicles.

[0015] In some embodiments of the present invention, musculoskeletal disorders include sprains, strains and tears of ligaments, tendons, muscles and cartilage, tendinitis, tenosynovitis, fibromyalgia, osteoarthritis, rheumatoid arthritis, intervertebral disc disease, polymyalgia rheumatica, bursitis, acute and chronic back pain, osteoporosis, carpal tunnel syndrome, De Quervain's disease, trigger finger, tennis elbow, rotator cuff, ganglion cyst, osteogenesis imperfecta, Duchenne muscular dystrophy, Hurler and Hunter syndrome, and combinations thereof. In one embodiment, the musculoskeletal disorder is osteoarthritis (OA).

[0016] In another embodiment of the present invention, the cardiac disease is myocardial infarction. In another embodiment of the present invention, the skin defect wound is a burn.

[0017] A further aspect of the present invention relates to a cell transplant composition comprising a cell transplant delivered with BiFP (a bifunctional peptide), where BiFP consists of a tissue targeting sequence and a cell binding motif sequence flanked by GPO repeats.

[0018] In some embodiments of the present invention, the cell graft composition induces the regeneration of neocartilage in OA knee joints. In some embodiments of the present invention, the cell graft composition induces corneal epithelial regeneration in areas of corneal epithelial defects. [Brief explanation of the drawings]

[0019] [Figure 1A] Circular dichroism (CD) spectra of peptides with representative secondary structures are shown. The triple helical structures of BiFP, collagen, and denatured collagen peptide were measured at 0.2 mg / ml. [Figure 1B] Figure 1 shows the particle size distribution of 0.2 mg / ml BiFP in PBS at 25°C as examined by dynamic light scattering (DLS). The data revealed a uniform size distribution (~1.914 nm). [Figure 2A] Analysis of hMSC binding to BiFP. hMSCs were labeled with Hoechst 33258 and incubated with FITC-labeled BiFP for 1 hour, followed by flow cytometry imaging on an Amnis Imaging Cytometer (left). Bar = 10 μm. Quantification of the percentage of BiFP-binding hMSCs (right). [Figure 2B] Analysis of hMSC binding to BiFP. hMSCs were labeled with Hoechst 33258 and incubated with FITC-labeled BiFP for 1 hour, followed by flow cytometry imaging on an Amnis Imaging Cytometer (left). Bar = 10 μm. Quantification of the percentage of BiFP-binding hMSCs (right). [Figure 3A]Measurement of viability of human mesenchymal stem cells (hMSCs) treated with the indicated concentrations of collagen XII-targeting peptide (Col12-TP) and BiFP. hMSCs were incubated with Col12-TP or BiFP peptide for 1 hour and stained with calcein-AM (green) and ethidium homodimer (magenta) before confocal microscopy (left). Bar = 50 µm. Quantification of the percentage of viable cells (right). [Figure 3B] 1 shows the growth curves of Col12-TP or BiFP-bound hMSCs analyzed by WST-1 proliferation assay. [Figure 3C] 1 shows Col12-TP or BiFP-bound hMSCs analyzed for mRNA levels indicated by real-time RT-PCR. [Figure 3D] Measurement of sulfated glycosaminoglycan (sGAG) production in Col12-TP- or BiFP-bound hMSCs at day 14 is shown. hMSCs were stained with 1,9-dimethylmethylene blue (DMB) dye and analyzed at OD525nm. Results were normalized by PicoGreen dsDNA quantification. Data are shown as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 were significant by Student's t-test (two groups) or analysis of variance (≥3 groups). [Figure 4A] FITC-labeled BiFP was intra-articularly injected into rat knee joints previously treated with papain / cysteine. 24 hours after injection, the entire joint capsule was removed, and the articular surface of the distal femur was observed under a two-photon microscope. Representative images of the collagen second harmonic generation (SHG) signal and BiFP fluorescence signal are shown (left). Quantification of the entire fluorescent area (right). [Figure 4B]DilC18 fluorescently labeled rat MSCs (rMSCs) without or with BiFP (BiFP+rMSCs) were intra-articularly injected into rat OA knee joints previously established with papain / cysteine. 24 hours after injection, the entire joint capsule was removed, and the articular surface of the distal femur was observed under a two-photon microscope. Representative images of the collagen second harmonic generation (SHG) signal and the rMSC fluorescence signal are shown (left). Quantification of the entire fluorescent area (right). [Figure 4C] Representative images of hematoxylin and eosin (H&E) staining of distal femurs from sham control or OA knee joints harvested 8 weeks after implantation with or without rMSCs delivered during BiFP, rMSCs, and BiFP (BiFP+rMSCs) are shown. [Figure 4D] Representative images of Safranin-O / Fast Green staining of distal femurs from sham control or OA knee joints harvested 8 weeks after implantation with or without rMSCs delivered in BiFP, rMSCs, and BiFP (BiFP+rMSCs) are shown. [Figure 4E] Analysis of OA severity by modified Mankin's score according to slides from H&E and Safranin-O / Fast Green staining is shown. Bar = 50 μm. Data are shown as mean ± standard deviation. *p<0.05, **p<0.01, and ****p<0.0001 were significant by Student's t-test (two groups) or one-way analysis of variance (≥3 groups). [Figure 5A] This is a schematic flowchart of the experimental design for regenerating multilayered articular cartilage with EGFP-expressing human MSCs, with or without delivery of BiFP (BiFP+hMSCs), in a papain / cysteine-induced rat osteoarthritis (OA) model. Rats were sacrificed at the indicated time points. Distal femurs from the knee joints were subjected to immunohistochemistry or immunofluorescence analysis. [Figure 5B] Immunohistochemistry for EGFP is shown. Positive signals are visualized as brown (DAB), and NC is a negative control without primary antibody. Bar = 50 μm. [Figure 5C]Immunofluorescence of EGFP, aggrecan, collagen 2, and collagen 12 was performed on rat OA cartilage treated with EGFP-hMSCs or BiFP-delivered EGFP-hMSCs to assess the expression of chondrogenic markers. Bar = 50 μm. [Figure 6A] This is a schematic flowchart of the experimental design for multilayered articular cartilage regeneration by EGFP-expressing rMSCs with or without BiFP (BiFP+rMSCs) in a papain / cysteine-induced rat osteoarthritis (OA) model. Rats were sacrificed at the indicated time points. The distal femurs of the knee joints were subjected to immunohistochemistry or immunofluorescence analysis. [Figure 6B] Immunohistochemistry for EGFP is shown, with positive signals visualized as brown (DAB). [Figure 6C] Double immunofluorescence of EGFP and the expression of chondrogenic markers such as aggrecan, collagen 2, or collagen 12 are shown. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Bar = 50 μm. [Figure 7A] This is a schematic flowchart of the experimental design for intervertebral disc (IVD) regeneration with PBS, BiFP, and EGFP-expressing hMSCs without or with delivery of BiFP (BiFP + hMSCs) in a rat intervertebral disc injury model induced by 20G needle puncture. Rats were sacrificed at the indicated time points. Rat tail tissues were subjected to X-ray, T2-weighted MRI, hematoxylin and eosin (H&E), Safranin O / Fast Green staining, immunohistochemistry, and immunofluorescence analysis. [Figure 7B] Shown is an X-ray image of the bone structure. [Figure 7C] T2-weighted MRI images of the nucleus pulposus (NP) of the coccygeal joint in rats that had received disc injury (the Sham group did not receive disc injury). After two weeks of treatment, specimens were taken for X-ray analysis. [Figure 7D] Representative images of H&E staining and Safranin-O / Fast Green staining of NP tissues are shown. [Figure 7E]Representative images of H&E staining and Safranin-O / Fast Green staining of NP tissues are shown. [Figure 7F] Immunohistochemistry for EGFP is shown, with positive signals visualized as brown (DAB). [Figure 7G] Double immunofluorescence of EGFP and expression of chondrogenic markers such as aggrecan or collagen 2 are shown. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Bar = 50 μm. [Figure 8A] FIG. 10 is a schematic flow chart of the experimental design for corneal epithelial regeneration with EGFP-expressing hMSCs without or with delivery in BiFP (BiFP+hMSCs) in a rat corneal epithelial injury model. [Figure 8B] Corneal epithelial defects (CEDs) induced by n-heptanol injury were treated with one drop (20 ml) of eye drops containing PBS, BiFP (40 μM), EGFP-hMSCs (105), or BiFP-delivered EGFP-hMSCs at the indicated times after treatment initiation, followed by fluorescein staining. Representative images of fluorescein-stained corneas are shown at the indicated time points (left). Quantification of the percentage of healed area was calculated using ImageJ Fiji software (right). *p<0.05, **p<0.01, and ***p<0.001 were significant by one-way ANOVA. [Figure 8C] Corneal histology in each group was detected by H&E staining on day 14. Bar = 50 μm. [Figure 8D] Immunohistochemistry for EGFP is shown. Positive signals are visualized as brown (DAB), NC is the negative control. Bar = 50 μm. DETAILED DESCRIPTION OF THE INVENTION

[0020] Other features and advantages of the present invention are further illustrated and explained in the following examples, which are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] Example 1: Preparation of a cell graft delivery system containing a bifunctional peptide (BiFP)

[0022] In this study, we synthesized a bifunctional peptide (BiFP) to avoid complex chemical processes and improve efficiency. A 42-amino acid residue peptide (BiFP) with the sequence GPOGPOGPOGPOGFOGERGPOGPOGPOGPODLQYWYPIWDTH, where O is hydroxyproline, was prepared by the Biomedical Translation Research Center (Taiwan) in PBS, pH 7.4, at 25°C. The triple helical structures of BiFP, collagen, and denatured collagen peptide were measured at 0.2 mg / ml each. The particle size distribution of BiFP at 0.2 mg / ml in PBS at 25°C was investigated by dynamic light scattering (DLS). Circular dichroism (CD) spectra confirmed that the peptide adopts a stable triple helical conformation in solution (Figure 1A). Size was assessed using dynamic light scattering: Malvern ZS90 Zetasizer (Malvern Instruments Corp, Malvern, UK). Dynamic light scattering data showed a uniform size distribution, with the predominant size being 1.914 nm in PBS (Figure 1B).

[0023] FITC-labeled BiFP or rhodamine-labeled collagen XII-targeting peptide (Col12-TP) was chemically synthesized at Biomertech (USA) or ABI (USA), and peptide binding was identified by confocal microscopy. Briefly, for FITC-BiFP or rhodamine-Col12-TP treatment, 1 x 10 hMSCs were incubated with 1 μM DilC18 for 10 min at 37°C, followed by incubation with 0, 2.5, 10, or 40 μM fluorescent peptide in a final 200 μl of PBS for 30 min at 37°C with gentle mixing every 10 min. Cells were counterstained with mounting solution containing DAPI. Peptide fluorescence was acquired using an ImageXpress Micro Confocal High Content Imaging system (Molecular Devices, Sunnyvale, CA, USA).

[0024] First, we demonstrate that Col12-TP cannot bind to hMSCs, whereas BiFP can bind to hMSCs in a dose-dependent manner. Imaging flow cytometry shows similar data to microscopy, further revealing that increasing the BiFP concentration from 2.5 to 40 μM results in an increased percentage of cells surrounded by FITC-conjugated BiFP (Figure 2A). Consistently, hMSCs do not express collagen XII, whereas chondrocyte cell line hiP cells express collagen XII and can bind to Col12-TP.

[0025] Additionally, another 42-amino acid peptide (BiFP) with the sequence GPOGPOGPOGPOGVMGFOGPOGPOGPOGPODLQYWYPIWDTH, where O is hydroxyproline, was also prepared. In this case, the GVMGFO sequence, a CLP containing a DDR-binding motif adjacent to GPO repeats, was incorporated into the collagen XII-targeting peptide. As shown in Figure 2B, BiFP can also bind to hMSCs. Similarly, imaging flow cytometry indicates that increasing the concentration of BiFP from 2.5 to 40 µM results in an increase in the percentage of cells surrounded by FITC-conjugated BiFP (Figure 2B). These data suggest that bifunctional peptides (BiFPs) containing the GVMGFO sequence, a DDR-binding motif, can also be designed to develop cell graft delivery systems for regenerative medicine.

[0026] Example 2: BiFP conjugation enhances viability and chondrogenic differentiation of MSCs.

[0027] For BiFP or Col12-TP treatment, 1 × 10 hMSCs were incubated in 200 μl of PBS containing 0, 2.5, 10, or 40 μM peptide for 30 min at 37°C with gentle mixing every 10 min. Cells were stained with calcein-AM (ThermoFisher) for live hMSCs and ethidium homodimer (Life Technologies) for dead hMSCs. Labeled hMSCs were visualized using an ImageXpress Micro Confocal High Content Imaging system (Molecular Devices, Sunnyvale, CA, USA). Three independent images from each group were counted and quantified as live hMSCs.

[0028] Compared to Col12-TP, human MSCs incubated with cultured BiFP exhibit increased cell viability (Figure 3A), cell proliferation (Figure 3B), and differentiation into chondrogenic cells, as indicated by increased expression of chondrogenic genes such as sox9, col2a1, and aggrecan after 1 week (Figure 3C) and enhanced glycosaminoglycan synthesis after 2 weeks (Figure 3D). Notably, the effects of BiFP on viability and proliferation are more pronounced at higher concentrations than at lower concentrations (Figure 3C). Collectively, these data suggest that BiFP conjugation dose-dependently promotes MSC viability, proliferation, and chondrogenic differentiation.

[0029] Example 3: Application of BiFP containing cell graft delivery system in OA regenerative medicine

[0030] Given that BiFP has the ability to induce chondrogenesis in OA cartilage and the integrin α2β1, it is likely that BiFP can induce MSCs to undergo chondrogenesis. Therefore, BiFP may be applicable to the delivery of human MSCs to the OA surface for OA regenerative medicine. To demonstrate the OA-specific targeting activity of BiFP, rhodamine-labeled BiFP was separately injected into the OA joints of a rat model with or without preincubation with collagen XII-blocking peptide, and two-photon microscopy observation of the fluorescence and second harmonic generation (SHG) signals was performed.

[0031] Surface-rendered 3D reconstruction images and transverse composite images showed clear red dots in BiFP-injected OA cartilage (Figure 4A). When probing type II collagen with SHG, the red dots were localized in areas without SHG signal, corresponding to the pericellular regions of OA cartilage. Conversely, no red dots were observed in OA cartilage injected with BiFP preincubated with a peptide that blocks collagen XII (Figure 4A).

[0032] MSCs were then labeled with Dil viability dye, incubated with or without BiFP, and subsequently injected intra-articularly into rat OA knee joints. When type II collagen was probed by SHG, red dots were observed on the articular surface of rat OA knees treated with MSCs incubated with BiFP, but not in rat OA knees treated with MSCs not incubated with BiFP (Figure 4B).

[0033] Furthermore, after incubation with BiFP, MSCs were immediately injected into the OA joints of a rat model, and the joints were subjected to histological examination 8 weeks after implantation. Histomorphometric analysis revealed successful induction of OA when comparing the OA group with the sham control group (Figure 4C, 4D). Furthermore, knee joints receiving MSCs delivered by BiFP showed clear cartilage formation and Safranin-O staining (Figure 4C, 4D). However, knee joints receiving MSCs without BiFP, Col12-TP alone, and BiFP alone still exhibited severe OA, exhibiting multiple cracks on the cartilage surface accompanied by loss of Safranin-O staining.

[0034] Quantification of OA severity using the modified Mankin score also revealed that the sham control group had a lower OA score than the OA group (Figure 4E).Similarly, OA treated with MSCs delivered in BiFP showed significantly improved modified Mankin scores compared with OA treated with MSCs not delivered in BiFP, Col12-TP alone, or BiFP alone (Figure 4E).

[0035] Human and rat MSCs were then lentivirally transduced with EGFP for long-term tracking, then delivered in BiFP and injected intra-articularly into the OA knee joints of rat osteoarthritis (OA) models once a week for 3 weeks (Figures 5A and 6A). Seven days after the third transplant, knee joints were harvested for cell fate assessment by histological evaluation of chondrogenic protein expression of the transplanted MSCs.

[0036] Because MSCs are immune-privileged, EGFP-human MSCs (EGFP-hMSCs) delivered in BiFP were intra-articularly injected into rat OA knee joints once a week for 3 weeks (Figure 5A), followed by histological evaluation of cartilage formation 1 week later. Remarkably, multiple layers of GFP+ neocartilage were observed arranged in three perpendicular blocks, with cells in the bottom and middle blocks possessing a mature chondrocyte-like spherical shape, and cells in the top block possessing an MSC-like fibroblastic morphology (Figure 5B). Furthermore, these cells were positive for aggrecan and collagen II (Figure 5C). Collectively, these data suggest that multiple injections of rat and human MSCs delivered in BiFP regenerate multiple layers of neocartilage.

[0037] Figure 6 shows the application of multiple intra-articular injections of bifunctional peptide (BiFP)-delivered rat MSCs (rMSCs) in regenerative medicine for osteoarthritis. Immunostaining reveals multiple layers of EGFP+, aggrecan+, and collagen II+ cells in knee joints receiving EGFP-rMSCs delivered in BiFP, but not in knee joints receiving EGFP-rMSCs not delivered in BiFP (Figure 6B). Furthermore, double immunofluorescence reveals colocalization of EGFP with aggrecan and collagen II (Figure 6C). Notably, multiple layers of collagen XII+ cells are observed in knee joints receiving EGFP-rMSCs without BiFP delivery, whereas only a single layer or sparse collagen XII+ cells are observed in knee joints receiving EGFP-rMSCs delivered in BiFP.

[0038] Example 4: Application of BiFP containing cell graft delivery system in intervertebral disc regeneration

[0039] Figure 7 shows the application of bifunctional peptide (BiFP)-delivered human MSCs (hMSCs) in intervertebral disc (IVD) regeneration. X-ray, T2-weighted MRI, and histological, histochemical, and immunofluorescence analyses reveal successful induction of IVD injury when comparing the PBS group with the sham control group (Figure 7B-7E). Furthermore, the nucleus pulposus receiving hMSCs delivered by BiFP maintained IVD height (Figure 7B) and demonstrated clear disc regeneration, as evidenced by T2-highly hydrated discs (Figure 7C) and Safranin-O-positive nucleus pulposus (Figure 7D). However, the MSCs not administered with BiFP, BiFP alone, and PBS groups still exhibited severe IVD damage, resulting in loss of disc height and T2-highly hydrated discs, as well as loss of Safranin-O staining. Immunostaining revealed multiple layers of EGPF+, aggrecan+, and collagen II+ cells in the nucleus pulposus that received EGFP-hMSCs delivered in BiFP, but not in the nucleus pulposus that received EGFP-hMSCs not delivered in BiFP (Figures 7F, 7G).

[0040] Example 5: Application of MSCs delivered in a BiFP containing cell graft delivery system for corneal epithelial regeneration

[0041] Collagen XII is expressed in various tissues, including the cornea. It is abundant in the stroma and the anteriorly located Bowman's layer, where it becomes exposed when the eye has a corneal epithelial defect. Corneal transplantation remains the primary method of visual rehabilitation when disease affects corneal transparency. Additionally, cadaveric corneal epithelial stem cells are transplanted with an amniotic membrane carrier for severe ocular surface disease and limbal dysfunction. However, all of these techniques rely on the availability of corneal donor tissue, which is a major limiting factor in developing countries.

[0042] To further confirm the usefulness of BiFP as a cell carrier and expand the application of BiFP-delivered MSCs in corneal epithelial defect regeneration, we utilized a rat model of severe corneal epithelial injury induced by repeated administration of n-heptanol. Measurement of the corneal defect area by sodium fluorescein staining revealed that administration of n-heptanol twice over four days resulted in failure of corneal epithelial healing after two weeks, suggesting that this is an important model of corneal epithelial injury (Figure 8A, flowchart).

[0043] Interestingly, corneas receiving hMSCs delivered with the BiFP containing cell graft delivery system showed no defect 1 week after corneal injury, whereas corneas receiving only hMSCs or BiFP showed significant defect even 2 weeks after injury (Figure 8B). Histomorphometric analysis confirmed the same result: hMSCs delivered with BiFP significantly improved corneal wound healing compared to hMSCs or BiFP alone (Figure 8C).

[0044] Immunohistochemistry showed that all neoepithelial cells were GFP+, indicating that hMSCs had successfully engrafted into the injured cornea and formed a neoepithelial cell layer (Figure 8D). Notably, the morphology of the neoepithelium formed by hMSCs resembled that of normal corneal epithelium, with flat cells in the upper layer and round cells in the lower layer (Figure 8D). These data suggest that BiFP acts as a cell carrier for the delivery of hMSCs in corneal epithelial regeneration.

[0045] In summary, we first synthesized a CLP containing a GFOGER or GVMGFO sequence flanking a GPO repeat sequence and incorporated it into a collagen XII-targeting peptide, thereby creating a bifunctional peptide (BiFP). The BiFP was then engineered to develop a cell graft delivery system for regeneration. For example, in cartilage regeneration, the cell graft delivery system may address the unmet need for damaged or degenerated articular cartilage and help repair or regenerate damaged or degenerated joints with a layer of newly grown chondrocytes. Furthermore, in corneal regeneration, hMSCs delivered by the cell graft delivery system significantly improved corneal wound healing in a rat model of corneal epithelial injury. Therefore, the cell graft delivery system could also be applied to regenerative medicine for degenerative disc disease, corneal injury in keratitis or dry eye syndrome, and other diseases involving collagen XII expression, such as myocardial infarction, burns, and alopecia.

Claims

1. a cell transplant and a BiFP (bifunctional peptide) that delivers the cell transplant to a target tissue; A cell graft delivery system, wherein the cell graft is bound to a BiFP consisting of a tissue targeting sequence and a cell binding motif sequence flanking GPO repeats.

2. The cell graft delivery system of claim 1 , wherein the tissue targeting sequence is an osteoarthritis (OA) or degenerative disc targeting sequence.

3. The cell graft delivery system of claim 1 , wherein the tissue targeting sequence is a collagen XII targeting sequence.

4. The cell graft delivery system of claim 1 , wherein the cell binding sequence is an integrin binding motif or a DDR binding motif.

5. The cell graft delivery system of claim 1, wherein the BiFP comprises a collagen XII targeting sequence, an integrin α2β1 binding motif sequence or a DDR binding motif sequence, and at least three copies of GPO adjacent to the integrin binding motif or the DDR binding motif.

6. The cell graft delivery system of claim 4 , wherein the integrin motif is an integrin α2β1 binding motif.

7. The cell graft delivery system of claim 6 , wherein the α2β1 binding motif has the sequence of GFOGER.

8. The cell graft delivery system of claim 4 , wherein the cell binding motif is a DDR binding motif.

9. The cell graft delivery system of claim 8 , wherein the DDR binding motif has the sequence of GVMGFO.

10. The cell graft delivery system of claim 1 , wherein the BiFP has the sequence GPOGPOGPOGPOGPOGFOGERGPOGPOGPOGPOGPODLQYWYPIWDTH.

11. The cell graft delivery system of claim 1 , wherein the BiFP has the sequence GPOGPOGPOGPOGPOGVMGFOGPOGPOGPOGPODLQYWYPIWDTH.

12. The cell graft delivery system of claim 1 , wherein the cell graft is selected from MSCs, musculoskeletal progenitor or differentiated cells, and corneal cells or their progenitor cells.

13. The cell graft delivery system of claim 1 , wherein the cell graft is an autologous MSC graft.

14. The cell graft delivery system of claim 1 , wherein the cell graft is an allogeneic MSC graft.

15. 1. A method for treating a disease involving expression of collagen XII, comprising: administering to a subject in need thereof a therapeutically effective amount of a cell graft delivered with a BiFP (bifunctional peptide); The BiFP comprises a tissue targeting sequence and a cell binding motif sequence flanking GPO repeats.

16. The method according to claim 15, wherein the disease involving the expression of collagen XII is a musculoskeletal disorder, sepsis or aseptic keratitis, dry eye syndrome, heart disease, skin defect wounds or a disease involving hair follicles.

17. 17. The method of claim 16, wherein the musculoskeletal disorders include sprains, strains and tears of ligaments, tendons, muscles and cartilage, tendinitis, tenosynovitis, fibromyalgia, osteoarthritis, rheumatoid arthritis, intervertebral disc disease, polymyalgia rheumatica, bursitis, acute and chronic back pain, osteoporosis, carpal tunnel syndrome, De Quervain's disease, trigger finger, tennis elbow, rotator cuff, ganglion cyst, osteogenesis imperfecta, Duchenne muscular dystrophy, Hurler and Hunter syndrome, and combinations thereof.

18. 17. The method of claim 16, wherein the musculoskeletal disorders include osteoarthritis (OA) and intervertebral disc disease (IVD).

19. 17. The method of claim 16, wherein the cardiac disease is myocardial infarction.

20. The method of claim 16, wherein the skin defect wound is a burn.

21. A cell transplant composition for regenerative medicine, comprising a cell transplant delivered with BiFP (bifunctional peptide), A cell transplant composition, wherein the BiFP comprises a tissue targeting sequence and a cell binding motif sequence flanking GPO repeats.

22. 22. The cell graft composition of claim 21, used to induce neocartilage regeneration in OA knee joints.

23. 22. The cell graft composition of claim 21, used to induce disc regeneration in an intervertebral disc with an IVD.

24. The cell graft composition of claim 21, which is used to induce corneal epithelial regeneration in a corneal defect area.

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