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The proliferation, osteogenic differentiation, and distribution patterns of stromal cells from rat bone marrow were investigated in a three-dimensional nonwoven fabric of polyethylene terephthalate fiber by the static, agitated, and stirred culture methods; stirring speeds were 10, 50, and 100 rpm in the stirred culture method. The culture method affected the time profile of proliferation and osteogenic differentiation of cells or their distribution in the fabric. The extent of cell proliferation and osteogenic differentiation became higher in order of the stirred at 100 rpm = the stirred at 50 rpmthe stirred at 10 rpmthe agitatedthe static methods. In addition, the cells were more uniformly proliferated in the fabric by the stirred culture method with time than they were proliferated in the fabric by other methods. The alkaline phosphatase (ALP) activity and calcium content were higher for cells cultured by the stirred culture method than those cultured by other methods. The total ALP activity, calcium content, and bone mineral density were higher for every stirred method than those for other methods. However, the distribution uniformity of cells differentiated was low irrespective of the culture method. It is concluded that the extent of proliferation and differentiation of cells or their distribution uniformity in the nonwoven fabrics was influenced by the culture method.

An ideal biomaterial for urethral reconstruction has not been developed. To create a urethral graft biomaterial with optimal biodegradability and biocompatibility, a copoly(L-lactide/epsilon-caprolactone) [P(LA/CL)] fabric tube was combined with a type I collagen sponge. The P(LA/CL) fibers were knitted into a vascular stent style (Type 1) or weaved into a mesh style (Type 2) to prepare P(LA/CL) tubes. The tubes were dipped in aqueous collagen solution and lyophilyzed to prepare the P(LA/CL)-collagen sponge graft. The grafts were applied to a 1.5-cm rabbit urethral defect (n = 14 for each condition), and tissue repair was evaluated using urethrographical, urethroscopical, and histological examination 1, 3, and 6 months after surgery. Although epithelialization was observed after 1 month in all Type 1 grafts, stenoses, fistulae, or stone formation was seen in 7 of the rabbits. In some cases, P(LA/CL) fibers prolapsed into the urethral lumen, causing stone formation. Only 3 rabbits survived for 6 months, and 2 of these had stenoses. For the Type 2 graft, all urethras were patent, without fistulae or stenoses, over the entire observation period. Histologically, urethral structure was disorganized for the Type 1 graft, whereas the urethral tissue on the Type 2 graft was slightly fibrotic but completely epithelialized and supported by a regenerated smooth muscle layer at 6 months. These findings suggest that creation of a scaffold suitable for urethral tissue regeneration will depend not only on the biomaterial composition, but also on the fabrication technique.

It is expected that mesenchymal stem cells (MSCs) will be a cell source for cardiac reconstruction because of their differentiation potential and ability to supply growth factors. However, poor viability at the transplanted site often hinders the therapeutic potential of MSCs. Here, in a trial designed to address this problem, a non-viral carrier of cationized polysaccharide is introduced for genetic engineering of MSCs. Spermine-introduced dextran of cationized polysaccharide (spermine-dextran) was internalized into MSCs by way of a sugar-recognizable receptor to enhance the expression level of plasmid deoxyribonucleic acid (DNA). When genetically engineered by the spermine-dextran complex with plasmid DNA of adrenomedullin (AM), MSCs secreted a large amount of AM, an anti-apoptotic and angiogenic peptide. Transplantation of AM gene-engineered MSCs improved cardiac function after myocardial infarction significantly more than MSCs alone. Thus, this genetic engineering technology using the non-viral spermine-dextran is a promising strategy to improve MSC therapy for ischemic heart disease.

This study is an investigation to evaluate in situ adipose tissue regeneration in fat pads. Gelatin microspheres with different water contents were prepared for the controlled release of basic fibroblast growth factor (bFGF). After a collagen sponge scaffold was incorporated by the microspheres containing 0, 0.01, 0.1, 1, and 10 microg of bFGF with or without syngeneic rat preadipocytes (1 x 10(5) cells/site) into a defect of rat fat pad, adipogenesis at the implanted site of scaffold was evaluated histologically. in situ formation of adipose tissue accompanied with angiogenesis was observed in the scaffold implanted with the microspheres containing 1.0 microg of bFGF, although the extent was less at the lower and higher bFGF doses. The in situ formation induced by the microspheres containing bFGF was significantly higher than that induced by free bFGF of the same dose. Adipogenesis was enhanced with time after implantation up to 4 weeks and thereafter leveled off. Such in situ adipogenesis was reproducibly induced by implantation of collagen scaffold incorporating gelatin microspheres containing 1 microg of bFGF, whereas addition of rat syngeneic preadipocytes did not promote the adipogenesis. The degradation of microspheres and the consequent FGF release became faster with an increase in the water content of gelatin microspheres. Less in situ formation of adipose tissue was observed at the lower water content of microspheres, which showed longer-term bFGF release. We conclude that combination of scaffold collagen with an appropriate controlled release of bFGF was essential to achieve the in situ formation of adipose tissue even without preadipocytes.

The objective of this study was to obtain fundamental knowledge about in vitro culture systems to enhance the proliferation and differentiation of mesenchymal stem cells (MSCs) in collagen sponge reinforced by the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously localized at PGA:collagen weight ratios of 0.67, 1.25, 2.5, and 5 was freezedried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treatment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. When rat MSCs were seeded into collagen sponge with or without PGA fiber incorporation, more attached cells were observed in collagen sponge incorporating PGA fiber than in collagen sponge without PGA fiber incorporation, irrespective of the PGA:collagen ratio. The proliferation and osteogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly influenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method became maximum at a flow rate of 0.2 mL/min, although they increased with culture time period. It may be concluded that appropriate perfusion conditions enable MSCs to positively improve the extent of proliferation and differentiation.

This study investigates the ability of platelet-rich plasma (PRP) combined with biomaterials to enhance in vivo bone-repairing activity. A biodegradable hydrogel was prepared from gelatin, which has an affinity for various growth factors. Rabbit PRP was conventionally prepared by blood centrifugation and dropped onto freeze-dried gelatin hydrogel to obtain gelatin hydrogel incorporating PRP. Gelatin hydrogel incorporating PRP was applied to a bone defect of rabbit ulna to evaluate bone formation at the defect in terms of soft X-ray and histological examinations. As controls, fibrin incorporating PRP, empty gelatin hydrogel, and free PRP were applied to the defect; in addition, defect without any application was examined. Successful bone regeneration was observed at bone defect treated with gelatin hydrogel incorporating PRP, in marked contrast to the control groups. When in contact with gelatin, growth factors, such as platelet-derived growth factor and transforming growth factor beta(1), were released from the PRP. PRP growth factors are immobilized in the hydrogel through physicochemical interaction with gelatin molecules. The immobilized growth factors are released from the hydrogel in concert with hydrogel degradation. It is likely that the gelatin hydrogel permitted the controlled release of bioactive growth factors, resulting in factor-induced promotion of bone regeneration.

We studied the effects of dexamethasone (Dex) and basic fibroblast growth factor (bFGF) on proliferation and differentiation of rat bone marrow stromal cells (RBMSCs), using three scaffolds: collagen sponge, poly(glycolic acid) (PGA)-collagen sponge, and PGA-collagen (UV) sponge. RBMSCs were seeded into the sponges, and cultured in primary medium, primary medium with Dex, and primary medium with bFGF and Dex. Three weeks after cultivation, we examined alkaline phosphatase (ALP) activity and cell number in the sponges, and also performed macroscopic, light microscopic, and scanning electron microscopic (SEM) observations. Collagen sponge shrank considerably, but PGA-collagen and PGA-collagen (UV) sponges maintained most of their original shape. PGA-collagen (UV) sponge supplemented with bFGF and Dex together had the highest ALP activity and cell number, followed by PGA-collagen sponge. Although collagen sponge showed cell proliferation only on the surface, the other two sponges showed cell proliferation in the interior. SEM showed the best cell attachment to PGA-collagen (UV) sponge in the presence of bFGF and Dex, followed by PGA-collagen sponge. In conclusion, PGA-collagen (UV) and PGA-collagen sponges proved to be much more useful as scaffolding for bone regeneration when combined with bFGF and Dex.

The initial attachment, proliferation, and osteogenic differentiation of stromal cells from human fat tissue were investigated in three-dimensional nonwoven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. The largest number of cells initially attached was observed in the nonwoven fabrics prepared from PET fiber with a diameter of 22.0 microm, irrespective of fabric porosity. The number of cells attached was larger and the cells were distributed more homogeneously in the fabrics by the agitated seeding method than by the static seeding method. The culture method depended on the time profile of cell proliferation. Cell proliferation improved in the following order: stirred (spinner flask) culture method > agitated culture method > static culture method. In addition, cells proliferated homogeneously in fabrics by the stirred culture method. When evaluated as a measurement of cell osteogenic differentiation, the activity of alkaline phosphatase (ALP) was not influenced by the diameter of fabrics. The static culture method tended to enable cells to enhance ALP activity, in contrast with the stirred and agitated culture methods. It is concluded that fabric fiber diameter and culture method greatly affected the proliferation and differentiation of cells in nonwoven fabrics.

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

The hair reconstitution assay is a useful system for studying cell-cell and epithelial-mesenchymal interaction. The current method consists of transplantation of both epidermal and dermal cells, using a silicone chamber placed on an athymic nude mouse. However, because of leakage and tilting of the grafted cells, the rate and area of hair growth vary depending on the chamber. We modified this method by using a collagen sponge as a scaffold and compared two types of collagen sponges, each having different tensile strengths. A conventional collagen sponge disturbed normal hair follicle formation; in contrast, a collagen sponge containing polyglycolic acid (PGA) fiber supported proper restructuring of skin and hair follicles. These data suggested the usefulness of PGA fiber-containing collagen sponges for hair reconstitution in research and clinical applications.

Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rats and seeded into a nonwoven fabric of polyethylene terephtalate (PET) by agitation and static methods. MSC attachment was investigated in terms of the number of cells attached to the fabric, their distribution inside the fabric, and cell damage. The number of MSCs attached was greater for the agitation seeding method than for the static seeding method. The higher the rotating speed in the agitation seeding method, the greater the number of cells attached. When the cell suspension was seeded into the fabric in culture medium volumes of 50 and 200 microL per well of the culture plate or per culture tube, the best cell attachment was observed for the tube culture group at the larger volume. These cells attached more homogeneously throughout the fabric in greater numbers than was the case for the other culture groups. It is possible that agitation of the cell suspension allows cells to infiltrate uniformly inside the fabric, resulting in a homogeneous distribution of the cells in the fabric. A biochemical study revealed that neither the agitation nor static seeding method damaged cells, irrespective of the medium volume and the type of culture vessel. We conclude that the agitation seeding method is a promising method by which to formulate a homogeneous construct of fabric and MSCs.

Tissue engineering is an emerging biomedical field intended to assist the regeneration of body tissue defects too large to self-repair as well as to substitute for the biological functions of damaged and injured organs by using cells with proliferative and differentiative potential. In addition to basic research on such cells, it is undoubtedly indispensable for successful tissue engineering to create an artificial environment enabling cells to induce tissue regeneration. Such an environment can be achieved by making use of a scaffold for cell proliferation and differentiation and for growth factors, as well as their combination. Growth factors are often required to promote tissue regeneration, as they can induce angiogenesis, which supplies oxygen and nutrients to cells transplanted for organ substitution to maintain their biological functions. However, the biological effects of growth factors cannot always be expected because of their poor in vivo stability, unless a drug delivery system is contrived. In this article, tissue regeneration based on the release of growth factors is reviewed to emphasize the significance of drug delivery systems in tissue engineering.

The present study was undertaken to investigate whether matrix metalloproteinase (MMP) functions to prevent the occurrence of destructive fibrosis in progressive renal disease. As a sustained release carrier of plasmid DNA, biodegradable hydrogels and microspheres were formulated from cationized gelatin prepared through aminization. Plasmid DNA was released from the cationized gelatin hydrogels as a result of hydrogel degradation. A plasmid DNA including a cytomegalovirus promoter and human recombinant MMP-1 gene (pCMV-MMP) was constructed. Gelatin microspheres incorporating pCMV-MMP as well as phosphate-buffered saline (PBS) with or without pCMV-MMP were injected into the renal subcapsule of C57BL/6 mice, which were intraperitoneally injected with streptozotocin (STZ) to induce diabetes 7 days after operation. The mice were killed 4 weeks after STZ injection to sample their blood and kidneys for biochemical and histological examinations. An immunofluorescence study confirmed that MMP protein was expressed around the renal tissue injected with gelatin microspheres incorporating pCMV-MMP. When applied with cationized gelatin microspheres incorporating pCMV-MMP, the mice showed a level of blood urea nitrogen significantly lower than that of other groups. A reduced content of collagen in the kidneys of mice administered gelatin microspheres incorporating pCMV-MMP was histologically observed. Further, the hydroxyproline assay revealed a significantly decreased content of hydroxyproline in kidney. We conclude that sustained release of MMP-1 gene is a promising prophylactic trial for kidney fibrolysis and dysfunction in the STZ-induced diabetic mouse model.

This article describes an investigation of collagen sponge mechanically reinforced through the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously dispersed at collagen:PGA weight ratios of 1.5, 0.8, 0.4, and 0.2 was freeze-dried, followed by dehydrothermal cross-linking to obtain collagen sponges incorporating PGA fiber to various extents. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. As expected, PGA fiber incorporation enabled the collagen sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly greater for any collagen sponge incorporating PGA fiber than for collagen sponge. The shrinkage of sponge after cell seeding was suppressed by fiber incorporation. It is possible that shrinkage suppression results in the superior cell attachment of sponge incorporating PGA fiber. After subcutaneous implantation into the backs of mice, the residual volume of collagen sponge incorporating PGA fiber was significant compared with that of collagen sponge and increased with a decrease in the collagen:PGA ratio. The greater number of cells infiltrated and deeper infiltration were observed for collagen sponge incorporating PGA fiber implanted subcutaneously. We conclude that the incorporation of PGA fiber is a simple and promising way to reinforce collagen sponge without impairing biocompatibility.

Controlled release of basic fibroblast growth factor (bFGF) from gelatin microspheres achieved de novo adipogenesis at the implanted site of a basement membrane extract (Matrigel). Following subcutaneous co-implantation of Matrigel and gelatin microspheres incorporating 0.1 microg of bFGF into the back of mice, adipose tissue was formed at the implanted site after 4 weeks postoperatively although the extent increased with implantation time. Formation of adipose tissue was significantly faster than the co-implantation of Matrigel, and 0.1 microg of free bFGF while a larger volume of the adipose tissue formed was retained 15 weeks later. When measured in Matrigel co-implanted with the gelatin microspheres incorporating bFGF, the number of cells infiltrated into Matrigel increased to a significantly high extent compared with the bFGF co-implantation. Matrigel alone was much less effective in inducing formation of adipose tissue. We conclude that gelatin microspheres incorporating bFGF enable Matrigel to efficiently induce de novo adipogenesis at the implanted site in respect to the formation rate and volume of adipose tissue.

This study is an investigation to evaluate how the controlled release of basic fibroblast growth factor (bFGF) affects the hair follicle growth of mice in different hair cycle stages: second anagen and second telogen. bFGF was incorporated into biodegradable gelatin hydrogels for its controlled release. After subcutaneous implantation of gelatin hydrogels incorporating 0, 0.7, 7, and 70 microg of bFGF or injection of 0 and 70 microg of free bFGF into the backs of mice, hair follicle growth was evaluated photometrically and histologically on the basis of three parameters: skin color of the reverse side of the implanted or injected site, skin thickness, and area occupied by hair follicle tissue. For mice in second anagen, the darkness of the reverse side of skin implanted with gelatin hydrogel incorporating 7 microg of bFGF was significantly higher than that of skin injected with 70 microg of bFGF 10 days after bFGF application. Implantation of gelatin hydrogel incorporating bFGF enabled the hair follicles to increase the area occupied in skin tissue to a significantly greater extent than in other groups, whereas no effect on skin thickness was observed. bFGF-free, empty gelatin hydrogels did not affect hair follicle growth. Moreover, hair shaft length was significantly elongated by gelatin hydrogel incorporating 7 microg of bFGF, in marked contrast to other agents. The skin of telogen mice receiving gelatin hydrogel incorporating 7 microg of bFGF did not show any change in darkness of reverse skin side or skin thickness, but a significant increase in the size of hair follicles 10 days later. These results indicate that the controlled release of bFGF positively affects the hair growth cycle of mice.

This study is a trial to promote repairing of the rabbit skull bone gap between an autologous bone flap and the intact bone with biodegradable gelatin microspheres containing transforming growth factor-beta1 (TGF-beta1). A 10-mm diameter bone defect was prepared in rabbit skulls by drilling out a bone flap of 6 mm in diameter. After a surrounding gap defect of 2 mm was created and treated with 0.5 microg of free TGF-beta1 and gelatin microspheres containing 0.5 microg of free TGF-beta1, the circular autologous bone flap was placed in the center. Significant bone healing at the gap defect was observed 3 weeks after implantation of the TGF-beta1-containing gelatin microspheres. The bone mineral density (BMD) was significantly higher than that of other experimental groups. On the contrary, when applied with free TGF-beta1, a fibrous tissue initially infiltrated into the gap defect, resulting in impairing bone healing. The tissue response was similar to that at the defect implanted with empty gelatin microspheres and TGF-beta1-free phosphate-buffered saline solution alone. There was more space in the gap-filling bone in the 16-week view than the 3-week view. It is possible that this was an intermediate step along the way toward normal healing and formation of cancellous bone. We conclude that gelatin microspheres containing TGF-beta1 show promise as an agent to promote bone regeneration of subcritical size defects between surgically positioned autologous bone flaps and surrounding host bone.

De novo adipogenesis at the implanted site of a basement membrane extract (Matrigel) was induced through controlled release of basic fibroblast growth factor (bFGF). bFGF was incorporated into biodegradable gelatin microspheres for its controlled release. When the mixture of Matrigel and bFGF-incorporated gelatin microspheres was implanted subcutaneously into the back of mice, a clearly visible fat pad was formed at the implanted site 6 weeks later. Histologic examination revealed that the de novo formation of adipose tissue accompanied with angiogenesis was observed in the implanted Matrigel at bFGF doses of 0.01, 0.1, and 1 microg/site, the lower and higher doses being less effective. The de novo formation induced by the bFGF-incorporated microspheres was significantly higher than that induced by free bFGF of the same dose. The mRNA of a lipogenesis marker protein, glycerophosphate dehydrogenase, was detected in the formed adipose tissues, biochemically indicating de novo adipogenesis. Free bFGF, the bFGF-incorporated gelatin microspheres, or Marigel alone and bFGF-free gelatin microspheres with or without Matrigel did not induce formation of adipose tissue. This de novo adipogenesis by mixture of Matrigel and the bFGF-incorporated gelatin microspheres will provide a new idea for tissue engineering of adipose tissue.

In vivo release of basic fibroblast growth factor (bFGF) from a biodegradable gelatin hydrogel carrier was compared with the in vivo degradation of hydrogel. When gelatin hydrogels incorporating 125I-labeled bFGF were implanted into the back subcutis of mice, the bFGF radioactivity remaining decreased with time and the retention period was prolonged with a decrease in the water content of the hydrogels. The lower the water content of 125I-labeled gelatin hydrogels, the faster both the weight of the hydrogels and the gelatin radioactivity remaining decreased with time. The decrement profile of bFGF remaining in hydrogels was correlated with that of hydrogel weight and gelatin radioactivity, irrespective of the water content. Subcutaneous implantation of bFGF-incorporating gelatin hydrogels into the mice induced significant neovascularization. The retention period of neovascularization became longer as the water content of the hydrogels decreased. To study the decrease of activity of bFGF when implanted, bFGF-incorporating hydrogels were placed in diffusion chamber and implanted in the mouse subcutis for certain periods of time. When hydrogels explanted from the mice were again implanted, significant neovascularization was still observed, indicating that most of the biological activity of bFGF was retained in the hydrogels. It was concluded that, in our hydrogel system, biologically active bFGF was released as a result of in vivo degradation of the hydrogel. The release profile was controllable by changing the water content of hydrogels.

We prepared a complex membrane consisting of two collagen membranes purified from human amnion and a polyglycolic acid (PGA) mesh. Using this membrane, we produced two types of sheets for pericardial replacement by glutaraldehyde cross-linking followed by succinylation [PGA-amnion (GA-S) sheet] or by dehydrothermal cross-linking followed by hyaluronic acid fixation [PGA-amnion (DHT-H) sheet]. We used these sheets to replace part of the pericardium in dogs, and the state of pericardial repair and regeneration were evaluated. As a control sheet, expanded polytetrafluoroethylene (EPTFE) with a thickness of 0.1 mm was used. After implantation of the PGA-amnion (DHT-H) sheet, soft connective tissue with abundant capillaries was observed, and its surface was covered with a mesothelial cell layer with many microvilli. The basement membrane was nearly normal. After implantation of the PGA-amnion (GA-S) sheet, dense connective tissue was observed, and its surface was covered with mesothelial cells. However, there ...

The objective of this study was to create a novel approach to promote bone induction through sustained release of growth factor from a 3-dimensional (3D) hybrid scaffold. Peptide-amphiphile (PA) was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. Collagen sponge was reinforced by incorporation of poly(glycolic acid) (PGA) fiber. A 3D network of nanofibers was formed by mixing basic fibroblast growth factor (bFGF) suspensions with dilute aqueous solutions of PA. A hybrid scaffold was fabricated by combination of self-assembled PA nanofibers and collagen sponge reinforced with incorporation of PGA fibers. The in vitro release profile of bFGF from hybrid scaffold was investigated, and ectopic bone formation induced by the released bFGF was assessed after subcutaneous implantation of hybrid scaffold into the backs of rats. Homogeneous bone formation was histologically observed throughout the hybrid scaffolds, in marked contrast to collagen sponge-incorporated bFGF. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high compared with collagen sponge incorporated with bFGF. The combination of bFGF incorporated in a collagen sponge self-assembled PA nanofiber hybrid scaffold is a promising procedure to improve bone regeneration.

The objective of this study was to investigate the feasibility of biodegradable gelatin hydrogels as the controlled-release carrier of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration at a skull defect of nonhuman primates. Hydrogels with 3 different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. A critical-sized defect (6 mm in diameter) was prepared at the skull bone of skeletally mature cynomolgus monkeys, and gelatin hydrogels incorporating various doses of BMP-2 were applied to the defects. When the bone regeneration was evaluated by soft radiography and bone mineral density (BMD) examinations, the gelatin hydrogel incorporating BMP-2 exhibited significantly higher osteoinduction activity than did an insoluble bone matrix that incorporated BMP-2 (one of the best osteoinduction systems), although the activity depended on the water content of hydrogels. BMD enhancement was highest for the gelatin hydrogel that had a water content of 97.8 wt% among all types of hydrogels. Moreover, the gelatin hydrogel enabled BMP-2 to induce the bone regeneration in nonhuman primates even at low doses. We conclude that the controlled release of BMP-2 for a certain time period was essential to inducing the osteoinductive potential of BMP-2.

The objective of the study was to test the hypothesis that platelet-rich plasma (PRP) enhances meniscal tissue regeneration in vitro and in vivo. In the in vitro study, monolayer meniscal cell cultures were prepared, and 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay and 5-bromo-2'-deoxyuridine assay were performed to assess proliferative behavior in the presence of PRP. Alcian blue assay was performed to assess extracellular matrix (ECM) synthesis. To detect the fibrocartilage-related messenger ribonucleic acid (mRNA) expressions, real-time polymerase chain reaction was performed. In the in vivo study, 1.5-mm-diameter full-thickness defects were created in the avascular region of rabbit meniscus. Gelatin hydrogel (GH) was used as the drug delivery system for PRP growth factors. The defects were filled as follows: Group A, GH with PRP; Group B, GH with platelet-poor plasma; Group C, GH only. Each group was evaluated histologically at 4, 8, and 12 weeks after surgery. PRP stimulated deoxyribonucleic acid synthesis and ECM synthesis (p

This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.

This article describes the prefabrication of a vascularized bone graft composed of autologous particulate cancellous bone and marrow (PCBM), a vessel bundle, and a biodegradable membrane. The PCBM was placed around the saphenous vessel bundle of rats and rolled with a biodegradable membrane of L-lactide-epsilon-caprolactone copolymer to prepare the prefabricated vascularized bone graft (group A). As controls, combinations of PCBM and membrane (group B), vessel bundle and membrane (group C), and PCBM and vessel bundle (group D) were prepared. A radiographic study revealed radio-opacity in the implantation site of group A 1 week later, in contrast to the other groups. Newly formed bone in the membrane roll was histologically confirmed, and neomicrovasculature circulating from the vessel bundle through the newly formed bone tissue was observed. The increase in alkaline phosphatase activity and osteocalcin content was significant for the group A preparation compared with the other groups. We concluded that the combination of autologous PCBM, a vessel bundle, and a biodegradable membrane was promising in the prefabrication of vascularized bone with good blood circulation.

This study is an investigation of the proliferation and differentiation of bone marrow stromal cells (BMSCs) on film substrates with different surface properties. Films of noncharged polymers with several water wettabilities; cell culture plates coated with collagen type I or IV, gelatin, or basic fibroblast growth factor (bFGF); and glass were used. BMSCs isolated from rat bone marrow were cultured on the various substrates in medium with dexamethasone [Dex(+)] or without dexamethasone [Dex(-)] to assess cell proliferation and differentiation. The number of proliferated BMSCs depended on the water wettability of substrates, although the cell number was greater in Dex(-) medium than in Dex(+) medium. Protein-coated substrates exhibited a high proliferation rate compared with noncoated substrates. Alkaline phosphatase (ALP) activity increased with increasing cell number, whereas ALP activity per cell correlated well with cell number. When cultured in Dex(+) medium containing bFGF or on culture plates coated with bFGF, BMSC proliferation tended toward enhancement with an increase in the amount of bFGF added in solution form, whereas it did not depend on the amount of bFGF in coated form. On the other hand, ALP activity and calcium content of BMSCs became maximal with bFGF coated at about 1 x 10(3) to 2 x 10(3) ng, in contrast to bFGF in solution form. Irrespective of the amount of bFGF, ALP activity and calcium content levels for bFGF in coated form were higher than for bFGF in solution form. It is concluded that the type of culture substrate and the manner of addition of bFGF affect the proliferation and differentiation of BMSCs.

To regenerate periodontal tissues, a sandwich membrane composed of a collagen sponge scaffold and gelatin microspheres containing basic fibroblast growth factor (bFGF) in a controlled-release system was developed according to the new concept of "in situ tissue engineering." A three-walled alveolar bone defect (3 x 4 x 4 mm) was made bilaterally in edentulous regions created mesially to the canines in both the maxilla and mandible of nine beagle dogs. A sandwich membrane with or without bFGF (100 microg) was implanted in each defect (each group, n = 18). During weeks 1, 2, and 4, histologic evaluation and histometric analyses were performed on three dogs. Throughout the 4 weeks, vascularization and osteogenesis were active only in the bFGF-treated group (p < 0.01). New cementum was formed (2.4 +/- 0.9 mm) on the exposed root surface at 4 weeks, and functional recovery of the periodontal ligament was indicated in part by the perpendicular orientation of regenerated collagen fibers. In the control group, epithelial downgrowth and root resorption occurred and the defects were filled with connective tissue. Thus, our sandwich membrane induced successful regeneration of the periodontal tissues in a short period of time.

A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.

This study evaluated the regenerative effects of platelet-rich plasma (PRP) for the degenerated intervertebral disc (IVD) in vivo. After induction of IVD degeneration in rabbits, we prepared PRP by centrifuging blood obtained from these rabbits. These PRP were injected into the nucleus pulposus (NP) of the degenerated IVDs after impregnation into gelatin hydrogel microspheres that can immobilize PRP growth factors physiochemically and release them in a sustained manner with the degradation of the microspheres. As controls, microspheres impregnated with phosphate-buffered saline (PBS) and PRP without microspheres were similarly injected. Histologically, notable progress in IVD degeneration with time courses was observed in the PBS control, PRP-only, and sham groups. In contrast, progress was remarkably suppressed over the 8-week period in the PRP group. Moreover, in immunohistochemistry, intense immunostaining for proteoglycan in the NP and inner layer of the annulus fibrosus was observed 8 weeks after administration of PRP-impregnated microspheres. Almost all microspheres were indistinct 8 weeks after the injection, and there were no apparent side effects in this study. Our results suggest that the combined administration of PRP and gelatin hydrogel microspheres into the IVD may be a promising therapeutic modality for IVD degeneration.

The objective of this study is to investigate the feasibility of a biodegradable hydrogel of gelatin as the controlled release carrier of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of bone regeneration at a segmental bone defect. Hydrogels with three different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Segmental critical-sized defects (20 mm) were created at the ulnar bone of skeletally mature New Zealand white rabbits, and gelatin hydrogels incorporating BMP-2 (17 microg/hydrogel) were implanted into the defects. When bone regeneration was evaluated by soft x-ray observation and bone mineral density (BMD) measurement, the gelatin hydrogels incorporating BMP- 2 exhibited significantly high osteoinduction activity compared with that of free BMP-2, although the activity depended on the water content of the hydrogels. Significantly higher BMD enhancement was observed in the gelatin hydrogel with a water content of 97.8 wt% than that with the lower or higher water content. We concluded that the biodegradable gelatin hydrogel is a promising controlled release carrier of BMP-2 for bone regeneration at the segmental bone defect.