Collagen matrix flap covalently bound to fibroblast growth factor and preparation method thereof

Abstract

The present invention discloses a collagen matrix flap covalently bound to fibroblast growth factor and a preparation method thereof. The collagen matrix flap is prepared by cross-linking oxidized sodium alginate (OSA) and bovine type I collagen. Subsequently, polyamide-amine dendrimer (PAMAM) is used as a connection carrier to covalently graft basic fibroblast growth factor (bFGF), thereby constructing a scaffold system capable of long-term sustained release of growth factors. The scaffold has good biocompatibility. Experiments have shown that the scaffold of the present invention exhibits clinical operability, superior biological activity to other growth factor-loaded scaffolds, and a more sustained growth factor release effect.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen-based scaffolds, and in particular to a collagen matrix flap covalently bound with fibroblast growth factor and a preparation method thereof. Background Art

[0002] Currently, periodontal soft tissue regeneration for gingival recession primarily involves coronally advanced flaps (CAFs) combined with subepithelial connective tissue grafts (SCTGs) or bioremedial materials. SCTGs are considered the standard treatment for gingival recession and are more effective in maintaining gingival margin stability than CAFs alone. However, autologous grafts must be harvested from the palate or maxillary tuberosity, increasing surgical time and postoperative discomfort, and potentially leading to complications such as donor site bleeding, infection, and sensory impairment. Furthermore, the limited amount of material available due to anatomical limitations, such as the greater palatine neurovascular bundle, can lead to insufficient grafts when treating gingival recession involving multiple teeth. Insufficient palatal mucosal thickness also increases the risk of postoperative complications. While the development of novel soft tissue replacement materials is a promising approach compared to SCTGs, currently available alternatives still have limitations in terms of volume stability and degradation rate. Furthermore, most soft tissue replacement materials currently on the market are imported and relatively expensive, placing a financial burden on patients.

[0003] Growth factors promote cell proliferation and tissue repair and regeneration, and are crucial elements of tissue engineering. Applications primarily include enriched natural growth factors, such as platelet-rich fibrin (PRF), various plasma- and growth factor-rich membranes, and recombinant human growth factors. PRF, as a graft replacement, demonstrates superior reduction in gingival recession compared to CAF alone; however, it significantly underperforms in terms of root coverage and keratinized tissue thickening compared to CAF combined with SCTG. Furthermore, its production process lacks standardized preparation and application specifications, and its quality is significantly affected by production equipment. Basic fibroblast growth factor (bFGF) has been shown to play an important role in angiogenesis, immune regulation, tissue regeneration, and wound healing. Studies have shown that combining recombinant human bFGF with scaffold materials in a canine gingival recession model can produce more new bone and connective tissue, and has the potential to improve root coverage. However, its effectiveness in alleviating gingival recession requires further verification. As described in patent CN101962409B, collagen and growth factors are directly combined together by covalent means. However, due to the limited amino groups of collagen, the problem of low growth factor loading rate still exists. Summary of the Invention

[0004] In response to the shortcomings of the above-mentioned background technology, the present invention aims to provide a collagen matrix flap covalently bound to fibroblast growth factor and a method for preparing the same. During the preparation of the collagen matrix flap, collagen is cross-linked with oxidized sodium alginate. After cross-linking, amino-rich polyamide-amine dendrimers are grafted onto the flap under the action of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to covalently load more basic fibroblast growth factor and enhance its sustained release effect.

[0005] The present invention is achieved by the following technical methods:

[0006] A method for preparing a collagen matrix flap covalently bound with fibroblast growth factor, the method comprising the following steps:

[0007] (1) Oxidized sodium alginate (OSA) was synthesized using the periodate oxidation method. 5 g of sodium alginate was dissolved in 200 ml of deionized water under magnetic stirring for a long time. 5 g of sodium periodate was then added and stirred at 25-37°C in the dark for 2-4 hours. 5 mL of ethylene glycol was then added to suspend the mixture and react for at least 0.5 hours. The reaction mixture was then dialyzed, frozen, and lyophilized to obtain OSA.

[0008] (2) OSA-Col scaffolds with a collagen concentration of 8 mg / ml and a mass ratio of OSA to collagen of 4-20:100 (w / w) were prepared as follows: first, bovine type I collagen was dissolved in 0.5 M acetic acid solution at 4°C and the pH was adjusted to neutral with 2 M NaOH solution. At the same time, OSA was dissolved in PBS (pH = 7.4). The OSA and Col solutions were mixed for 30 minutes, and the amino groups of the collagen and the aldehyde groups of OSA were cross-linked via Schiff base, and then centrifuged to remove bubbles. Subsequently, each 2.5 ml of the mixture solution was transferred to a fixed-size mold with a length of 18 mm, a width of 23 mm, and a thickness of 6 mm. The mixture was incubated in an oven at 37°C for more than 4 hours for self-assembly, and then frozen at -20°C overnight. After freeze-drying, a collagen-cross-linked oxidized natural polysaccharide scaffold (abbreviated as OSA-Col scaffold) was obtained. Among them, too low collagen concentration and OSA / Col mass ratio will affect the mechanical strength of the gel, while too high collagen concentration and OSA / Col mass ratio will form an overly dense pore structure, affecting cell migration and ingrowth and the excretion of cell metabolites during soft tissue healing. Therefore, the optimal collagen concentration is determined to be 8 mg / ml and the OSA to Col mass ratio is 16:100 in this application.

[0009] (3) The previously obtained fixed-size OSA-Col scaffold was immersed in a 50 mM MES buffer solution (pH = 5.5) containing a polyamidoamine dendrimer (PAMAM) at a concentration of 5-20 mg / ml and bFGF at a concentration of 50-150 ng / ml. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (at a concentration of 5 mM) and N-hydroxysuccinimide (NHS) (at a concentration of 5 mM) were then added for cross-linking. The pH was adjusted to 5.5 using NaOH or HCl, and the resulting solution was incubated at 4°C for 24 hours. Subsequently, the solution was washed with deionized water for 30 minutes and freeze-dried at -20°C to obtain the collagen matrix flap covalently bound to fibroblast growth factor (i.e., P-OSA-Col@bFGF scaffold, referred to as POC@bFGF).

[0010] Among them, too low a PAMAM concentration cannot effectively increase the number of collagen amino groups and thus improve the grafting efficiency, while too high a PAMAM concentration may also produce steric hindrance and polycationic effects, which will instead reduce the grafting and sustained-release efficiency of the growth factor. In addition, too low a growth factor concentration does not play a biological role in promoting cell proliferation, and too high a growth factor concentration does not bring about a higher cell proliferation effect, but may instead bring about cytotoxic side effects. Therefore, during the preparation of the scaffold, the PAMAM concentration and the growth factor concentration need to be strictly controlled within an appropriate concentration range. In addition, the present application also determines that the optimal PAMAM concentration for biological scaffold grafting is 10 mg / ml, and the optimal concentration of bFGF is 100 ng / ml.

[0011] The present invention also provides a collagen matrix flap prepared by the above method.

[0012] The inventive principle of the present invention is:

[0013] To address the problem of excessively rapid metabolism associated with direct use of growth factors, the present invention designs materials to encapsulate and immobilize growth factors in the form of scaffolds, constructing a growth factor delivery system to achieve long-term sustained release. Type I collagen can enhance its mechanical properties by cross-linking and oxidizing sodium alginate. The grafting efficiency of growth factors can be further increased by grafting polyamide-amine dendrimers. Growth factors can be covalently grafted while retaining collagen activity, and sustained release in vivo can be achieved through material degradation. Therefore, the present invention proposes grafting fibroblast growth factor onto materials with good biocompatibility and moderate degradation rates to construct a cell-responsive soft tissue replacement material to promote gingival tissue regeneration and repair.

[0014] The present invention has the following advantages:

[0015] (1) Compared with the scaffold physically mixed with growth factors (POC + bFGF), the scaffold of the present invention has a higher grafting efficiency for fibroblast growth factor, and the grafting efficiency is highest when the PAMAM concentration is 10 mg / ml;

[0016] (2) Compared with the scaffold physically mixed with growth factors, the scaffold of the present invention can achieve a sustained release effect of up to 28 days, and the sustained release effect is best when the PAMAM concentration is 10 mg / ml;

[0017] (3) Compared with the biological scaffold without fibroblast growth factor, the scaffold loaded with fibroblast growth factor of the present invention has stronger cell proliferation promoting activity when the growth factor concentration is 100 ng / ml;

[0018] (4) Compared with the non-oxidative natural polysaccharide cross-linked biological scaffold (Col), the scaffold of the present invention has good mechanical properties and better clinical operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the preparation principle of the stent of the present invention;

[0020] Figure 2 Fourier transform infrared spectra of the stent prepared by the method of the present invention and the stent of the control group;

[0021] Figure 3 The microscopic morphology and pore size distribution of the scaffold prepared by the method of the present invention and the scaffold of the control group;

[0022] Figure 4 The swelling ratio of the scaffold prepared by the method of the present invention and the scaffold of the control group;

[0023] Figure 5 The amino concentration of the PAMAM scaffold grafted with different concentrations according to the method of the present invention;

[0024] Figure 6 is the growth factor loading rate of bFGF grafted onto the scaffolds in each group;

[0025] Figure 7 This is the 28-day growth factor release curve of each group of scaffolds;

[0026] Figure 8 The shear and cyclic compression properties of the scaffolds prepared by the method of the present invention and the control scaffolds;

[0027] Figure 9 CCK-8 cell proliferation activity of the scaffolds prepared by the method of the present invention and the control scaffolds;

[0028] Figure 10 These are the cell live-dead staining results of the scaffolds prepared using the method of the present invention and the scaffolds of the control group. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below with reference to examples.

[0030] A method for preparing a collagen matrix flap covalently bound to fibroblast growth factor, the preparation principle is as follows Figure 1 , specifically including the following steps:

[0031] (1) Oxidized sodium alginate (OSA) was synthesized using the periodate oxidation method. 5 g of sodium alginate was dissolved in 200 ml of deionized water under magnetic stirring for a long time. 5 g of sodium periodate was then added and stirred at 25-37°C in the dark for at least 4 h. 5 mL of ethylene glycol was then added to suspend the mixture and react for 0.5 h. The reaction mixture was then dialyzed, frozen, and lyophilized to obtain OSA.

[0032] (2) OSA-Col scaffolds with a final collagen concentration of 8 mg / ml and an OSA / Col mass ratio of 4-20:100 (w / w) were prepared. Bovine type I collagen was first dissolved in 0.5 M acetic acid solution at 4°C and the pH was adjusted to neutral with 2 M NaOH solution. At the same time, OSA was dissolved in PBS (pH = 7.4). The OSA and Col solutions were mixed for 30 minutes and then centrifuged to remove bubbles. Subsequently, each 2.5 ml of the mixture solution was transferred to a fixed-size mold with a length of 18 mm, a width of 23 mm, and a thickness of 6 mm. The mixture was incubated in an oven at 37°C for 4 hours and then frozen at -20°C overnight. After freeze-drying, the OSA-Col scaffold was obtained.

[0033] (3) To prepare the scaffolds, the previously obtained fixed-size OSA-Col scaffolds were immersed in 10 ml of a 50 mM MES buffer solution (pH = 5.5) containing 5-20 mg / ml PAMAM, 50-150 ng / ml bFGF, 5 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 5 mM N-hydroxysuccinimide (NHS). The pH was adjusted to 5.5 using NaOH or HCl, and the resulting solution was incubated at 4°C for 24 hours. Then, the scaffolds were washed with deionized water for 30 minutes, frozen at -20°C overnight, and freeze-dried to obtain POC@bFGF scaffolds with different PAMAM concentrations.

[0034] Additionally, as shown in Table 1, collagen scaffolds containing or without OSA were immersed in 10 ml of MES buffer (pH 5.5) containing varying concentrations of PAMAM, bFGF, and various components to produce various bioscaffolds. The pH was adjusted to 5.5, and the resulting solutions were incubated at 4°C for 24 hours. The scaffolds were then washed with deionized water for 30 minutes, frozen at -20°C, and freeze-dried to produce experimental scaffolds containing varying concentrations of PAMAM and bFGF, as well as other control scaffolds.

[0035] Table 1 Compositions of the scaffolds in the experimental and control groups

[0036]

[0037] The performance of various scaffolds prepared in the above examples and control groups was tested. The test results demonstrated that the preparation process of the scaffolds of the present invention does not destroy the original structural conformation of collagen, and that the resulting scaffolds possess key properties for treating gingival recession (such as pore size distribution, swelling ratio, and mechanical strength). Furthermore, the successful introduction of PAMAM increased the grafting and sustained release efficiency of basic fibroblast growth factor, enhancing the scaffold's bioactivity.

[0038] Specifically, the performance test results of each group of brackets are as follows:

[0039] (1) Fourier transform infrared spectroscopy (FTIR-ATR): The analysis showed that both the Col sample and the bFGF-loaded sample retained the integrity of the key amide bands, including 3315 cm -1 The amide A band at 2936 cm -1 The amide B band at 1655 cm -1 Amide I band at 1545 cm -1 The amide II band at 1246 cm -1 The amide III band at the position was not affected. Figure 2 , indicating that chemical cross-linking and the introduction of growth factors as well as different concentrations of PAMAM did not destroy the structural integrity of collagen.

[0040] (2) Microstructural features were examined using a scanning electron microscope. Each single layer sample with a thickness of 3 mm was sliced ​​and its cross section was observed. A pore size analysis was performed, and the results were as follows: Figure 3 As shown, the average pore size of each group is above 150 μm, among which the pore size distribution of the P10OC@bFGF group is the most uniform.

[0041] (3) The morphological stability of the stent in the oral environment is crucial, and swelling is an important factor affecting its volume change. Figure 4 It can be seen that the Col@bFGF group and the P10OC@bFGF group reached swelling equilibrium at 30 minutes first, while the other groups reached equilibrium after 3 hours, and the swelling rate of the P10OC@bFGF group was the smallest.

[0042] (4) Since PAMAM is rich in amino groups, the ninhydrin experiment shows that the amino content increases significantly with the increase of PAMAM concentration. Figure 5 As shown in the figure, compared with the Col group, the amino groups of samples with PAMAM concentrations of 5, 10, 15, and 20 mg / ml increased by 2, 3, 4, and 6 times, respectively. This proves the successful introduction of PAMAM and its amino groups.

[0043] (5) By Figure 6 As can be seen, the bFGF loading rate exceeded 80% in all groups, likely due to the physical encapsulation effect of the scaffold's porous structure and the electrostatic interaction between the scaffold and bFGF. The PAMAM-loaded groups showed significantly higher bFGF loading rates than the unloaded PAMAM-loaded groups (Col@bFGF, OSA-Col@bFGF) and the scaffold physically mixed with growth factors (POC+bFGF). This suggests that the amino groups introduced by PAMAM additionally enhance bFGF loading efficiency through covalent bonding.

[0044] (6) By Figure 7 Compared to a scaffold physically loaded with growth factors (POC+bFGF), the PAMAM-grafted scaffold achieved sustained release for up to 28 days. A similar growth factor release trend was observed on day 7, with the POC+bFGF group achieving a maximum release of 82.5%. Over the next 28 days, the P10OC@bFGF group had the lowest release rate, at only 41%. This indicates that a PAMAM concentration of 10 mg / ml provides optimal sustained release efficiency for the bioscaffold.

[0045] (7) By Figure 8 As can be seen, the PAMAM-grafted scaffold exhibited superior clinical operability compared to all other bioscaffold groups. Suture shear testing demonstrated excellent knotting performance, and the P10OC@bFGF group withstood significantly greater shear forces in a wet state than the other groups. Furthermore, the P10OC@bFGF group demonstrated the best compressive strength in compression cycling testing. These important mechanical properties demonstrate its excellent mechanical properties and clinical operability. Based on these performance characteristics, the optimal concentration of PAMAM for bioscaffold loading was determined to be 10 mg / ml.

[0046] (8) By Figure 9 It can be seen that the CCK-8 experiment showed that compared with the scaffolds not loaded with bFGF (Col, OSA-Col, POC), the groups with different growth factor concentrations (50, 100, 150 ng / ml) had higher proliferation activity of gingival fibroblasts, among which the POC@bFGF10 group had the strongest ability to promote cell proliferation.

[0047] (9) By Figure 10 As can be seen, the number of dead cells in each group was negligible, indicating that none of the scaffolds were cytotoxic. The number of live cells in the groups with added growth factors was significantly higher than that in the scaffolds without bFGF (Col, OSA-Col, POC). The POC@bFGF10 group had the highest number of live cells, indicating that the bioscaffold with the corresponding growth factor concentration had the best cell proliferation capacity for gingival fibroblasts.

Claims

1. A method for preparing a collagen matrix flap covalently bound to fibroblast growth factor, characterized in that: The following steps are involved: Step 1: First, collagen is dissolved at 4°C, and the collagen is mixed with oxidized natural polysaccharides, wherein the oxidized natural polysaccharide is oxidized sodium alginate, and the ratio of the oxidized sodium alginate to the collagen is 16:100 (w / w). The oxidized sodium alginate and collagen are mixed for 30 minutes, and the amino groups of the collagen and the aldehyde groups of the oxidized natural polysaccharide are cross-linked by Schiff base. At the same time, the collagen self-assembles to form a gel. The self-assembly is performed by incubating the mixture of the collagen and the oxidized natural polysaccharide at 37°C for 4 hours for self-assembly, and freeze-drying to obtain a collagen cross-linked oxidized natural polysaccharide scaffold; Step 2: Immerse the collagen cross-linked oxidized natural polysaccharide scaffold in an MES buffer solution containing a polyamidoamine dendrimer and growth factors, add a cross-linking agent and perform a cross-linking reaction at 4°C. After the reaction is completed, freeze-dry again to obtain a collagen matrix flap covalently bound to fibroblast growth factor; The concentration of the polyamidoamine dendrimer is 10 mg / ml; The growth factor is basic fibroblast growth factor, with a concentration of 100 ng / ml; The cross-linking agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking agent.

2. A collagen matrix flap covalently bound with fibroblast growth factor prepared by the method of claim 1.

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