Preparation And Study Of Collagen Scaffold Combined With Platelet-rich Plasma

Chapter1Preparation and Physico-Chemical Property of Rat Tail Type I CollagenObjective:To evaluate the feasibility of rat tail collagen using in biomaterials, the absorbance value and molecular weight of collagen solution, microstructure of collagen film, cytotoxicity and biocompatibility of collagen were investigated.Method:The fresh tendons were extracted from SD rat tails. The peritendineum and blood vessel were carefully removed. After immerging in0.1%benzalkonium chloride solution for10min and washing by0.9%saline, the tendons were cut into small fragments. Then, the tendon fragments were lysed in0.5M ethanoic acid solution for4days at4℃.The mixture was discontinuously pendulated during the process of solvation. The mixed solution was centrifuged for25min at high-speed ratio and4℃.The powders of sodium chloride were added into collected supernate and the mixture was stired continuously. When the flocculation occurred, the mixture was centrifuged once again. The sediments were collected and washed by distilled water for3times. Following dissolve the sediments by ethanoic acid solution, the PH value of collagen solution was adjusted to7by NaOH solution. The depurated collagen solution could be obtained after the process of dialysis. A slight amount of solution was left for SDS-PAGE analysis and spectral analysis. The remaining proportion was freeze-dried. Spectral analysis was determined by a ultraviolet-uisible spectrophotometer using rat tail type I collagen (Sigma) as standard. The molecular weight and isoforms distribution were detected by SDS-PAGE analysis. The structural feature of freeze-dried films was observed by SEM. The cytotoxicity of samples was assessed by the MTT colorimetric assay. Collagen solutions and films were sterilized by gamma irradiation and immersed in low glucose Dulbecco’s modified Eagle’s medium (L-DMEM) at37℃for48h. L-929mouse fibroblast cells were seeded in96well plates at a density of4.0×104cells/well in0.1ml L-DMEM supplemented with10%fetal bovine serum (FBS), cultured at37℃and5vol%CO2for24h and then treated by leaching liquor of samples. The cells were treated with a normal culture media as control group. After1,3, and7days of culture, the medium was then removed and the cells were washed once with PBS, then20μl of0.5%MTT solution was added to each well followed by incubation for4h at37℃and5vol%CO2. Subsequently, the MTT solution was removed and150μl DMSO was added to each well. The96well plates were placed on a shaker for10min and the optical density (OD) value of each well was measured at490nm using an ELISA reader.Results:The extracted collagen solution was translucent and viscous. It turned to be more transparent and be similar to gelatum after purification. The color of collagen film was white and the sponge-like structure of collagen film possessed elasticity and extensibility. Compared with the standerd rat tail type I collagen, the maximum absorption peak of the extracted collagen solution was about299nm, while the former was300nm. SDS-PAGE analysis indicates that the extracted and purified collagen from rat tail tendons is mainly of type I, which was characterized by the presence of two alpha chains (al,a2) and a beta component. Their molecular weights were approximately120,110and210kDa, which resembles control. The fiber-and lattice-like structure of collagen film with pore size ranged from100to250μm could be observed by SEM. The collagen liquors could be coagulated after irradiation while the collagen film had nothing to change. MTT assay showed that the cytotoxic grades were0or1at1st,3th and7th day. There is no statistical significance when compared with the control (p>0.05).Conclusion:The purified type I collagen can be extracted from rat tails. Our results suggest that both collagen solution and film have no cytotoxicity to L-929fibroblast cells. Due to their excellent cell compatibility, they can be used in tissue engineering. Chapter2The Effects of Different Crossing-Linking Conditions of Genipin on Type Ⅰ Collagen Scaffolds:An In-Vitro EvaluationObjective:The purpose of this chapter was to analyze the properties of fabricating rat tail type Ⅰ collagen scaffolds cross-linked with genipin under different conditions and to assess the feasibility of these scaffolds using in osteochondral tissue engineering. The morphologies, mechanical properties, cross-linking degree, swelling ratio, in vitro degradation, biocompatibility and cytotoxicity of the scaffolds were evaluated.Method:The collagen films were lysed in0.5M ethanoic acid solution and adjusted PH value to7. They were transferred into48-well microplates (800μL/well) and freeze-dried. Scaffolds were divided into nine groups and cross-linked by immersion in10ml of PBS containing different concentrations (0.1,0.3and0.5wt%) of genipin for24h at different temperatures (4℃,20℃,37℃). The non-cross-linked scaffolds were set as control. After being cross-linked, scaffolds of different groups were washed with distilled water to remove any residual genipin that might still be present. Afterwards, the3D scaffolds were freeze-dried again and sealed into plastic bags. After general observation, the matrices were fixed by mutual conductive adhesive tape on copper stubs and covered with gold using a sputter coater. The morphology of the scaffolds prepared was observed by a scanning electron microscope. The mechanical test was carried out using a material testing machine (Instron5540, USA) by compression in the vertical direction at a deformation rate of1.5mm/min until failure at20℃. The compressive strength was calculated by Q=Fmax/S, where Fmax is the maximum load on the load-deformation curve and S is the cross-sectional area of each sample. The dry scaffolds were weighed (w0) and then hydrated in PBS for3h at room temperature. After carefully removing the excess surface water with filter paper, the wet scaffolds were weighed (w) again. The swelling ratio of the scaffolds was defined as the wet weight increase (w-w0) to the initial weight (w0). The non-cross-linked collagen sponges were set as the controls. The cross-linking degree (CD) of the different groups was determined by ninhydrin assay. The scaffolds were weighed and a7mg sample from each different group was heated to100℃in a water bath with4ml NHN solution for20min. The solution was then cooled down to20℃, diluted with5ml50%isopropanol, and the optical absorbance of the solution at570nm was measured with a spectrophometer using glycine at various concentrations (1.0-5.0mg/mL) as standard. The equation was used for testing the cross-linking degree of the sample as follows: CD=(NHN reactive eamine)fresh-NHN reactive eamine)fixed/(NHN reactive eamine)fresh×100%. The ’fresh’ element means the mole fraction of free NH2in non-cross-linked samples while ’fixed’ indicate the mole fraction of free NH2remaining in cross-linked samples. The biodegradability of the type Ⅰ collagen scaffolds was determined by incubating each sample in2mL PBS (pH7.4) containing 200μg collagenase type I (sigma, USA) at4℃for12h. Afterwards, the reaction was discontinued by adding200μL0.2Methylenediaminetetraacetic acid (EDTA) and cooling the commixture in an ice bath immediately. The supernatant of the mixture was hydrolyzed in6M Hcl at110℃for24h. The pigment of solution was eliminated by absorbite and filtrated by filtration membrane. The mixture of2ml filtrate,2ml citrate buffer solution and2ml0.05M chloramine T were oxidated at room temperature for10min. After that,2ml perchloric acid solutions were added in the mixture above.10min later,2ml paradimethylaminobenzaldehyde solutions were added in the mixed solution for coloration at65℃for10min. The ultraviolet spectroscopy absorbance of hydroxyproline was examined by a spectrophometer using hydroxyproline at various concentrations (0-5.0mg/mL) as standard. The biodegradation degree is defined as the proportion of hydroxyproline content in the cross-linked samples to that in non-cross-linked ones. The extract liquids of different groups were prepared respectively at a concentration of1.25cm2of surface area of scaffolds per milliliter of L-DMEM medium and incubated at37℃for48h. The L-DMEM medium was reffered as the control group. The cytotoxicity was determined using MTT assay. The primary chondrocytes were isolated from the joint cartilage and xiphoid process of SD rat (3week) by enzymatic digestion. The second passage chondrocytes suspension were seeded on per scaffold (3×105cells/scaffold). Cell morphology and adherence is evaluated on the cross-linked collagen scaffolds, at1day and3day, by SEM.Results:The general shape of cross-linked collagen scaffolds were not changed obviously. The collagen scaffolds cross-linked with genipin produced blue pigment. The color appearance of collagen scaffolds changed with the different genipin cross-linking conditions. It seemed that the higher concentration of GP and cross-linking temperature increased the intensity of blue. The cross-section of the cross-linked structures was analyzed by scanning electron microscopy (SEM). All scaffolds presented a three-dimensional interconnected porous structure. Non-cross-linked collagen scaffold with pore size ranged from100to250μm presented the fiber-and lattice-like structure. However, the morphologies of the genipin cross-linked scaffolds undergo a sheet-like structural transition. Although the pore sizes have not been changed markedly, it seems that the sheet-like framework closely aligned with the higher genipin concentration and cross-linking temperature. In addition, the fibers of non-cross-linked collagen scaffold are totally not seen in all cross-linked groups. The compressive strength augments greatly with the increased genipin concentration and cross-linking temperature compared with control (p<0.05). The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked)(P<0.05). The swelling ratios of the scaffolds which in0.3%37℃,0.5%20℃and0.5%37℃groups were significantly lower than that of in0.1%4℃(P<0.05). The cross-linking degree ranged from6.90to26.48%when cross-linked by0.1%genipin. The cross-linking degree of scaffolds which in0.3%and0.5%groups were obviously higher than that of in0.1%groups(P <0.05). MTT assay showed that the ratios of cell proliferation were above80%and cytotoxic grades were0or1at1st,3th and7th day. After being treated with genipin for24h, the anti-degradation ability of collagen scaffolds increased remarkably. There is statistical difference between0.3%20℃,0.3%37,0.5%groups and0.1%4℃and0.3%4℃group. The chondrocytes maintain a round shape in day1indicating that they just adhere to the scaffold. At day3, the cells have adhered to the scaffold closely. So, these scaffolds can be used in osteochondral tissue engineering.Conclusion:Genipin could be used in cross-linking type I collagen scaffolds. Although the producted blue pigment, the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin were satisfactory. Based on our data, the optimization of process conditions demonstrated that successful cross-linking with genipin could be achieved at0.3%genipin concentrations and37℃. The scaffolds possessed exceptional swelling ratios, biodegradation degrees, cross-linking degrees, compressive strength and lower cytotoxicity under this condition. Chapter3Preparation and Study on Growth Factor Release of COL/PRP ScaffoldsObjective:The extracted PRP were activated respectively by thrombin and type I collagen solution. Then, the col/prp scaffolds could be obtained by freeze-drying the mixture of activated PRP and type I collagen solution according to a certain proportion. To evaluate the feasibility of these scaffolds using in osteochondral tissue engineering, the analysis of PRP fraction, structural feature, mechanics strength, cytotoxicity of scaffolds and contents of growth factor release were investigated.Method:6-8ml of blood could be extracted from each SD rat heart. The contents of leucocytes, erythrocytes and platelets of whole blood were measured by an automated animal blood counter. After the blood samples were centrifugated at1500rmp for10min, the blood separated into three phases:platelet-poor plasma (top), the platelet-rich plasma containing leucocytes and platelets (middle), and erythrocytes (bottom). The top and middle layers were transferred to new tubes and centrifuged again at3000rmp for10min. The supernatant plasma was discarded, and the remaining500μL of plasma containing precipitated platelets was blended evenly and designated as PRP. Care was taken to remove erythrocytes by a lml syringe to minimize their interference as far as possible. Samples of PRP were analyzed again by an automated animal blood counter and stored at-80℃. The collagen films were lysed in0.5M ethanoic acid solution and adjusted PH value to7. Samples of PRP were thawed at room temperature and poured into48-well microplates (300μL/well). The mixture of300μL PRP+30μL thrombin (30IU) was defined as thrombin-activated group,300μL PRP+300μL collagen solution was collagen-activated group,600μL collagen was blank group.10min later,300μL collagen solutions were added in each well of thrombin group. The mixed solutions were stired uniformly and freeze-dried. The morphology of the scaffolds prepared was observed by a scanning electron microscope. The mechanical test was carried out using a material testing machine. The rest of scaffolds were sterilized by ethylene oxide. The extract liquids of different groups were prepared respectively according to the method in chapter2. The L-DMEM medium was reffered as the control group. The cytotoxicity was determined using MTT assay. The sterilized scaffolds in thrombin-activated group and ollagen-activated group were placed into penicillin phials.2ml of L-DMEM medium was poured into each phial and incubated at37℃and5vol%CO2. Interval release of TGF-β1, PDGF, FGF and VEGF from each scaffold was measured at1,4,7and10days. At each time point, one ml of media was aspirated from around each sample and replaced with one ml of fresh media. Media samples were stored in1.8ml cryovials in a-80℃freezer until all samples were collected. Concentrations of rat PDGF, TGF-β1, FGF and VEGF were determined using the commercially available Quantikine colorimetric sandwich ELISA kits.Results:The color of freeze-dried COL/PRP scaffolds was pink. Though it possesed comparative elasticity, the texture of COL/PRP scaffolds was more brittle than the COL ones when compared with the latter. The COL/PRP scaffolds were analyzed by scanning electron microscopy (SEM) and presented a three-dimensional interconnected porous structure with pore size ranged from50to80μm. The platelet content in PRP was about3.91±0.98times more than in whole blood after centrifugation. While leukocytes and erythrocytes content was about1.11±0.40and 0.31±0.20times more than in whole blood respectively. The compressive strength of COL/PRP scaffolds was comparable with COL scaffolds. There was no statistical significance between them (p>0.05). There was no cytotoxicity to L-929fibroblasts in COL/PRP groups. The ratio of cellular proliferation was higher than control at day1and3. The results of four growth factors released by COL/PRP scaffolds as follow:The content of TGF-β1was highest at day4. Although the content fell off at day7and10, the higher concentration could be maintained in each group. The content of FGF and PDGF fell off with time gradually. The concentration of two growth factors in thrombin group was higher than collagen one at each time point. As far as VEGF, the content went up from day1to day4and fell off at day7. While at day10, it went up once again. The concentration of VEGF in two groups was similar at each time point.Conclusion:The COL/PRP scaffolds possessed exceptional porosity, strength and no cytotoxicity. The four growth factors released from scaffolds could be maintained for a long period and higher concentration. Thus, it is possible that these scaffolds can be used in osteochondral tissue engineering. Chapte4Study on Rat Calvarial Bone Regeneration of COL/PRP ScaffoldsObjective:The purpose of this chapter was to analyze the bone regeneration of COL/PRP scaffolds by implanting them into rat calvarial defect and to assess the feasibility of these scaffolds using in osteochondral tissue engineering. The histology and Micro-CT scanning were carried out.Method:The thrombin-activated COL/PRP scaffolds and collagen scaffolds were fabricated according to chapter3and sterilized by ethylene oxide.48Sprague-Dawley rats weighing250g were divided into three groups randomly: COL/PRP group, collagen group and blank group.0.3%phenobarbital sodium was intraperitoneally injected to induce general anesthesia. The rat cranium was exposed under sterile conditions. Five-millimeter-diameter trephine defects were created unilaterally in the calvaria of Sprague-Dawley rats under constant irrigation and with care to avoid injury to the underlying dura. Each defect was flushed with saline solution to remove bone debris. The scaffolds were implanted into calvarial defect. The blank group had no scaffolds.4Animals in each group were sacrificed after4,8and12weeks respectively, and calvaria were harvested for high-resolution microCT analysis. Decalcified samples were embedded in paraffin, and sections were stained with hematoxylin-eosin (H&E) and Masson-Goldner trichrome stain. The information of tissue repair in defect region was observed by light microscope.Results:The diet and activity of animals were normal after surgery. The incisions were healed primarily without red swelling, exudate and suppuration. There was no difference between COL/PRP group and collagen group at4weeks. The defect area was covered by a thin membrane. It seemed that the membrane in blank group was more transparent. The membrane became thick in COL/PRP group at8weeks, while collagen and blank group was thinner than former in turn. This situation was more obvious at12weeks. There was no tissue necrosis in all groups. The bone regeneration could not be seen at the defect region in COL/PRP group at4weeks. The defect region was filled mainly by fibrous tissue with abundant fibroblasts and new capillaries. The same thing happened in collagen group. The undegradative collagen fiber and slight new capillaries could be seen in fibrous connective tissue. The new bone could not be found at the edge of host bone. The fibrous connective tissue in blank group was thinner than former groups. New capillaries and bone regeneration could not be seen in the fibrous tissue and at the edge of host bone respectively. The immature lamellar bone occurred in COL/PRP group and filled in defect area together with fibrous connective tissue. Like4weeks, there was no bone regeneration in collagen or blank group. The defect only was filled with fibrous connective tissue. At12weeks, the defect area could be filled with much mature lamellar bone and fibrous connective tissue in COL/PRP group. Slight new bone and a great quantity of fibrous connective tissue could be seen at the defect in the collagen group. Bone regeneration formed at the edge of host bone. The bluish collagen fiber stained by Masson staining could be seen in the fibrous connective tissue. In blank group, the defect area was filled with fibrous connective tissue which thickness had not been augmented. There was no bone regeneration around host bone or in the fibrous tissue. The images of bone regeneration were obversed by high-resolution microCT. The defect region in the COL/PRP group had not been decreased at4weeks. Slight bone repair happened at the edge of host bone. While at8weeks, the defect area became irregular and the newly mineralized bone with higher density could be seen at the extension area of host bone. The most defect area was covered by bew bone and became further decreased. The density of bew bone was closed to the host bone. The ossification indexes of defect area could be evaluated by software of the microCT. Most indexes increased gradually with times. This indicated that COL/PRP scaffolds possesed the ability of osteoinduction. Although the slight ossification could be found in collagen group and defect area became decreased with times, but this trend was not obvious when compared with COL/PRP group. The collagen maybe possesed the ability of osteoinduction as the ossification indexes of8and12weeks were higher than4weeks. There was no difference in blank group at different times. Although the slight ossification could be found at the edge of host bone, the whole defect area changed unobviously. The images of microCT showed that the repair ability of blank group was worst in spite of the ossification indexes of8 and12weeks were higher than4weeks.Conclusion:The COL/PRP scaffolds possesed favourable ability of osteoinduction and repairing bone defect.