Xenopus laevis as a novel model to study long bone critical-size defect repair by growth factor-mediated regeneration

We used the tarsus of an adult Xenopus laevis frog as an in vivo load-bearing model to study the regeneration of critical-size defects (CSD) in long bones. We found the CSD for this bone to be about 35% of the tarsus length. To promote regeneration, we implanted biocompatible 1,6 hexanediol diacrylate scaffolds soaked with bone morphogenetic proteins-4 and vascular endothelial growth factors. In contrast to studies that use scaffolds as templates for bone formation, we used scaffolds as a growth factor delivery vehicle to promote cartilage-to-bone regeneration. Defects in control frogs were filled with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation and evaluated using micro-computed tomography and histology. In frogs treated with growth factor-loaded scaffolds, we observed a cartilage-to-bone regeneration in the skeletal defect. Five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months, these processes were well advanced, and extensive ossification was observed in 6-month samples. In contrast, the defects in control frogs showed only formation of fibrous scar tissue. This study demonstrates the utility of a Xenopus model system for tissue engineering research and that the normal in vivo mechanism of endochondral bone development and fracture repair can be mimicked in the repair of CSD with scaffolds used as growth factor delivery mechanisms.     

Cross-species comparison of ectopic bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) scaffolds

Material-induced bone formation reported in canine, bovid, suid, and primate species does not often occur in lagomorph or rodent models. In this study, we test biphasic calcium phosphate and hydroxyapatite- induced bone formation in subcutaneous pockets of mice and intramuscular pockets in rats, rabbits, and dogs. All scaffolds are of similar size, and all animals were sacrificed at 90 days post-implantation. In dogs (N = 8), all implants showed bone formation with significantly more bone formed in biphasic calcium phosphates (30 +/- 6%, N = 8) as compared to hydroxyapatite (14 +/- 5%, N = 8) (p = 0.003). Hydroxyapatite implants did not induce bone formation in mice, rats, or rabbits. Biphasic calcium phosphate induced bone in 6 of 8 scaffolds implanted in 4 rabbits and 3 of 16 scaffolds implanted in 16 mice, whereas it did not induce bone formation in any of the 8 rats. The results presented herein suggest that the incidence of material-induced bone formation varies with animal species and is related to the implant material used.     

Bone formation in transforming growth factor beta-1-coated porous poly(propylene fumarate) scaffolds

This study determined the bone growth into pretreated poly(propylene fumarate) (PPF) scaffolds implanted into a subcritical size, rabbit cranial defect. PPF scaffolds were constructed by using a photocrosslinking-porogen leaching technique. These scaffolds were then either prewetted (PPF-Pw), treated with RF glow-discharge (PPF-Gd), coated with fibronectin (PPF-Fn), or coated with rhTGF-beta1 (PPF-TGF-beta1). One of each scaffold type was then placed into the cranium of nine rabbits. The rabbits were sacrificed after 8 weeks, and the scaffolds were retrieved for histological analysis. The most bone formation was present in the PPF-TGF-beta1 implants; the newly formed bone had a trabecular appearance together with bone marrow-like tissue. Little or no bone formation was observed in implants without rhTGF-beta1. These histological findings were confirmed by image analysis. Bone surface area, bone area percentage, pore fill percentage, and pore area percentage were significantly higher in the rhTGF-beta1-coated implants than in the noncoated implants. No statistical difference was seen between the PPF-Fn, PPF-Pw, or PPF-Gd scaffolds for these parameters. Quadruple fluorochrome labeling showed that in PPF-TGF-beta1 implants bone formation mainly started in the interior of a pore and proceeded toward the scaffold. We conclude that (a) PPF-TGF-beta1 scaffolds can indeed adequately induce bone formation in porous PPF, and (b) PPF scaffolds prepared by the photocrosslinking-porogen leaching technique are good candidates for the creation of bone graft substitutes.     

In vivo mesenchymal cell recruitment by a scaffold loaded with transforming growth factor beta1 and the potential for in situ chondrogenesis

The objectives of this study were (1) to develop a biphasic implant made of a bioresorbable polymeric scaffold in combination with TGF-beta1-loaded fibrin glue for tissue-engineering applications, and (2) to determine whether the implant made of a polycaprolactone (PCL) scaffold and TGF-beta1-loaded fibrin glue could recruit mesenchymal cells and induce the process of cartilage formation when implanted in ectopic sites. Twenty-four 6-month-old New Zealand White rabbits were used. Scaffolds loaded with various doses of TGF-beta1 in fibrin glue were implanted subcutaneously, intramuscularly, and subperiosteally. The rabbits were killed and implants were removed at 2, 4, and 6 weeks postoperatively. The specimens were subjected to various staining techniques for histological analysis. Light microscopic examination of all specimens revealed that the entire pore space of the scaffolds was filled with various tissues in each group. The entire volume of the scaffolds in the groups loaded with TGF-beta1 and implanted intramuscularly and subcutaneously was populated with mesenchymal cells surrounded with an abundant extracellular matrix and blood vessels. The scaffold loaded with TGF-beta1 and implanted subperiosteally was found to be richly populated with chondrocytes at 2 and 4 weeks and immature bone formation was identified at 6 weeks. We conclude that scaffolds loaded with TGF-beta1 can successfully recruit mesenchymal cells and that chondrogenesis occurred when this construct was implanted subperiosteally.     

Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation

Three different types of porous hydroxyapatite with pore sizes of 100-200 micrometer in diameter-porous particles of hydroxyapatite (PPHAP), porous blocks of hydroxyapatite (PBHAP), and honeycomb-shaped hydroxyapatite (HCHAP)-were compared in terms of their abilities to induce osteogenesis when implanted subcutaneously with recombinant human BMP-2 into rats and extracted at 1, 2, 3, and 4 weeks. Histologically, direct bone formation occurred in PPHAP and PBHAP while only endochondral ossification took place in HCHAP. Interestingly, cartilage in the central zones and bone in the orifice zones of the tunnels of the HCHAP were observed at 2 weeks. After 3 weeks, the cartilage disappeared and bone formation occurred throughout the inner surface of the tunnels of the HCHAP, always leaving space for capillaries within the tunnels. Alkaline phosphatase activity and osteocalcin content were the highest in HCHAP among the three hydroxyapatite implants. These results clearly indicate that BMP-induced bone formation is highly dependent on the geometry of the carrier, which provides feasible structural factors for vascularization.     

In vivo bone generation via the endochondral pathway on three-dimensional electrospun fibers

A new concept of generating bone tissue via the endochondral route might be superior to the standard intramembranous ossification approach. To implement the endochondral approach, suitable scaffolds are required to provide a three-dimensional (3-D) substrate for cell population and differentiation, and eventually for the generation of osteochondral tissue. Therefore, a novel wet-electrospinning system, using ethanol as the collecting medium, was exploited in this study to fabricate a cotton-like poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffold that consisted of a very loose and uncompressed accumulation of fibers. Rat bone marrow cells were seeded on these scaffolds and chondrogenically differentiated in vitro for 4 weeks followed by subcutaneous implantation in vivo for 8 weeks. Cell pellets were used as a control. A glycosaminoglycan assay and Safranin O staining showed that the cells infiltrated throughout the scaffolds and deposited an abundant cartilage matrix after in vitro chondrogenic priming. Histological analysis of the in vivo samples revealed extensive new bone formation through the remodeling of the cartilage template. In conclusion, using the wet-electrospinning method, we are able to create a 3-D scaffold in which bone tissue can be formed via the endochondral pathway. This system can be easily processed for various assays and histological analysis. Consequently, it is more efficient than the traditional cell pellets as a tool to study endochondral bone formation for tissue engineering purposes.     

Biodegradable magnesium scaffolds: Part 1: appropriate inflammatory response

Current tissue engineering strategies focus on the replacement of pathologically altered tissues by the transplantation of cells in combination with supportive biocompatible scaffolds. Scaffolds for tissue engineering strategies in musculoskeletal research require an appropriate mechanical stability. In recent studies, considerable attention has thus been given to magnesium alloys as biodegradable implants. The aim of this study was to characterize the biocompatibility of magnesium scaffolds by the inflammatory host response. Open porous scaffolds made of the magnesium alloy AZ91D were implanted into the distal femur condyle of rabbits and were compared to autologous bone, which was transplanted into the contralateral condyle in a 3 and 6 months follow-up group. After 3 months, magnesium scaffolds were already largely degraded and most of the original magnesium alloy has disappeared. Concomitantly, a fibrous capsule enclosed the operation site. Histological analysis revealed that the magnesium scaffolds caused no significant harm to their neighboring tissues. This study shows that even fast degrading magnesium scaffolds show a good biocompatibility and react in vivo with an appropriate inflammatory host response. Magnesium alloy based implants are therefore a very promising approach in the development of mechanically suitable and open porous scaffolds for the replacement of subchondral bone in cartilage tissue engineering.     

Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vivo

Optimal repair of chondral defects is likely to require both a suitable population of chondrogenic cells and a biodegradable matrix to provide a space-filling structural support during the early stages of cartilage formation. This study examined the ability of chondrocytes to support cartilage formation when incorporated into biodegradable scaffolds constructed from copolymers (PLG) of polylactic acid (PLA) and polyglycolic acid (PGA) and implanted in the calf muscle of nude mice. Scaffolds were fabricated to be more hydrophilic (PLG-H) or were reinforced with 10% PGA fibers (PLG-FR), increasing the stiffness of the implant by 20-fold. Confluent primary cultures of rat costochondral resting zone chondrocytes (RC) were loaded into PLG-H foams and implanted intramuscularly. To determine if growth factor pretreatment could modulate the ability of the cells to form new cartilage, RC cells were pretreated with recombinant human platelet derived growth factor-BB IPDGF-BB) for 4 or 24 h prior to implantation. To assess whether scaffold material properties could affect the ability of chondrogenic cells to form cartilage, RC cells were also loaded into PLG-FR scaffolds. To determine if the scaffolds or treatment with PDGF-BB affected the rate of chondrogenesis, tissue at the implant site was harvested at four and eight weeks post-operatively, fixed, decalcified and embedded in paraffin. Sections were obtained along the transverse plane of the lower leg, stained with haematoxylin and eosin, and then assessed by morphometric analysis for area of cartilage, area of residual implant, and area of fibrous connective tissue formation (fibrosis). Whether or not the cartilage contained hypertrophic cells was also assessed. The amount of residual implant did not change with time in any of the implanted tissues. The area occupied by PLG-FR implants was greater than that occupied by PLG-H implants at both time points. All implants were surrounded by fibrous connective tissue, whether they were seeded with RC cells or not. The amount of fibrosis was reduced at eight weeks for both implant types. When RC cells were present, the amount of fibrosis was less than seen in cell-free scaffolds. Pretreatment with PDGF-BB caused a slightly greater degree of fibrosis at four weeks than was seen if untreated cells were used in the implants. However, at eight weeks, if the cells had been exposed to PDGF-BB for 24 h, fibrosis was comparable to that seen associated with cell-free scaffolds. The cells supported an equivalent area of cartilage formation in both scaffolds. PDGF-BB caused a time-dependent decrease in cartilage formation at four weeks, but at eight weeks, there was a marked increase in cartilage formation in PDGF-BB-treated cells that was greatest in cells exposed for 4 h compared to those exposed for 24 h. Moreover, PDGF-BB decreased the formation of hypertrophic cells. The results indicate that in this model, RC cells produce cartilage; pretreatment of the RC cells with PDGF-BB promotes retention of a hyaline-like chondrogenic phenotype; and the material properties of the implant do not negatively impact on the ability of the cells to support chondrogenesis.     

Three-dimensional polycaprolactone scaffold-conjugated bone morphogenetic protein-2 promotes cartilage regeneration from primary chondrocytes in vitro and in vivo without accelerated endochondral ossification

As articular cartilage is avascular, and mature chondrocytes do not proliferate, cartilage lesions have a limited capacity for regeneration after severe damage. The treatment of such damage has been challenging due to the limited availability of autologous healthy cartilage and lengthy and expensive cell isolation and expansion procedures. Hence, the use of bone morphogenetic protein-2 (BMP-2), a potent regulator of chondrogenic expression, has received considerable attention in cartilage and osteochondral tissue engineering. However, the exact role of BMP-2 in cartilage repair has been postulated to promote both cartilage formation and subsequent cartilage degradation through hypertrophy and endochondral ossification. Furthermore, it is likely that the manner in which BMP-2 is presented to chondrocytes will influence the physiologic pathway (repair vs. degeneration). This study investigates the relative influence of BMP-2 on cartilage matrix and potential subsequent bone matrix production using primary chondrocytes seeded on designed 3D polycaprolactone (PCL) scaffolds with chemically conjugated BMP-2. The results show that chemically conjugated BMP-2 PCL scaffolds can promote significantly greater cartilage regeneration from seeded chondrocytes both in vitro and in vivo compared with untreated scaffolds. Furthermore, our results demonstrate that the conjugated BMP-2 does not particularly accelerate endochondral ossification even in a readily permissible and highly vascular in vivo environment compared with untreated PCL scaffolds. This study not only reveals the potential use of the BMP-2 conjugation delivery method for enhanced cartilage tissue formation but also gives new insights for the effects of conjugated BMP-2 on cartilage regeneration and osteochondral ossification.     

Ectopic bone formation in rats: the importance of the carrier

Much research has been done to develop the ideal bone graft substitute (BGS). One approach to develop this ideal BGS is the use of growth factors, but for this approach osteoprogenitor cells are needed at the site of reconstruction. An alternative is a cell-based approach, where enough cells are provided to form bone in a carrier material. In previous studies of our group, titanium (Ti) carriers have been used, because of the excellent mechanical properties and the bone-compatibility of this material. On the other hand, calcium phosphate (CaP) ceramics are known for their excellent osteoconductivity. The aim of this study is to investigate the influence of the carrier in a cell-based bone regeneration approach, whereby we hypothesize that CaP-ceramic implants will induce more bone formation than Ti-fiber implants, in the same animal model as our previous experiment. Ti-fiber mesh implants and ceramic implants were seeded with rat bone marrow cells (RBM) and implanted subcutaneously. Histological analysis after one, three and six weeks showed differences in the way of bone formation in the two groups: bone appeared to grow from the center to the periphery of the implant in the titanium group, while bone formation in the ceramic group occurred through the whole implant. Histomorphometrical analysis after one week showed very limited bone formation for both the titanium and ceramic group. At three weeks, the amount of bone formation was increased till about 10% for the titanium group and 18% for the ceramic group. No significant difference between the two groups could be observed. In the six week group, the bone formation was 6% (Ti) and 23% (CaP), respectively (P < 0.001). Further, bone formation started earlier in the CaP-ceramic scaffolds than in the Ti scaffolds. Our hypothesis could be confirmed: ceramic implants induce more bone formation than titanium implants.     

Biotechnological approach for systemic delivery of membrane Receptor Activator of NF-κB Ligand (RANKL) active domain into the circulation

Deficiency of Receptor Activator of NF-κB Ligand (RANKL) prevents osteoclast formation causing osteopetrosis. RANKL is a membrane-bound protein cleaved into active soluble (s)RANKL by metalloproteinase 14 (MMP14). We created a bio-device that harbors primary osteoblasts, cultured on 3D hydroxyapatite scaffolds carrying immobilized MMP14 catalytic domain. Scaffolds were sealed in diffusion chambers and implanted in RANKL-deficient mice. Mice received 1 or 2 diffusion chambers, once or twice and were sacrificed after 1 or 2 months from implants. A progressive increase of body weight was observed in the implanted groups. Histological sections of tibias of non-implanted mice were negative for the osteoclast marker Tartrate-Resistant Acid Phosphatase (TRAcP), consistent with the lack of osteoclasts. In contrast, tibias excised from implanted mice showed TRAcP-positive cells in the bone marrow and on the bone surface, these latter morphologically similar to mature osteoclasts. In mice implanted with 4 diffusion chambers total, we noted the highest number and size of TRAcP-positive cells, with quantifiable eroded bone surface and significant reduction of trabecular bone volume. These data demonstrate that our bio-device delivers effective sRANKL, inducing osteoclastogenesis in RANKL-deficient mice, supporting the feasibility of an innovative experimental strategy to treat systemic cytokine deficiencies.     

Biphasic calcium phosphate: a scaffold for growth plate chondrocyte maturation

While skeletal development can occur by either intramembranous or endochondral bone formation, all current tissue engineering approaches for bone repair and regeneration try to mimic intramembranous ossification. In this study, we propose to create an in vitro cartilage template as the transient model for in vivo endochondral bone formation. The goals of this study are to (1) establish a method of growing chondrocytes in a well-characterized macroporous biphasic calcium phosphate (MBCP) scaffold and (2) induce maturation of chondrocytes grown in the MBCP scaffold. Chondrocytes isolated from chick embryonic tibia were grown on MBCP particles and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Chondrocytes were observed to attach and proliferate on the MBCP scaffold. The thickness of the chondrocyte and extracellular matrix layer increased in the presence of the retinoid. Alkaline phosphatase activity and expression, proteoglycans synthesis, cbfa1 and type I collagen mRNA levels also increased in the presence of retinoic acid. These results demonstrated for the first time the proliferation, maturation of chondrocytes, and matrix deposition on MBCP, suggesting the potential for such scaffold in tissue engineering via the endochondral bone formation mechanism.     

Biodegradable magnesium scaffolds: Part II: peri-implant bone remodeling

In this study, histomorphometrical parameters of the peri-implant bone remodeling around degrading open-porous scaffolds made of magnesium alloy AZ91D were investigated and compared with the peri-implant bone remodeling around an autologous bone transplant in the contralateral side in a rabbit model after 3 and 6 months. Osteoblast activity was displayed by collagen I (alpha 2) mRNA in situ hybridization. Major scaffold degradation was completed within 3 months after implantation showing no osteolysis around the scaffolds, both after 3 and 6 months. Enhanced formation of unmineralized extracellular matrix and an enhanced mineral apposition rate adjacent to the degrading magnesium scaffolds were accompanied by an increased osteoclastic bone surface, which resulted in higher bone mass and a tendency to a more mature trabecular bone structure around the magnesium scaffolds compared to the control. These results show that even fast-degrading magnesium scaffolds induce extended peri-implant bone remodeling with a good biocompatibility. In summary, this study shows that degrading magnesium scaffolds promote both bone formation and resorption in a rabbit model and are therefore very promising candidates for the development of novel implants in musculoskeletal surgery.     

Human osteoprogenitor bone formation using encapsulated bone morphogenetic protein 2 in porous polymer scaffolds

The ability to deliver, over time, biologically active osteogenic growth factors by means of designed scaffolds to sites of tissue regeneration offers tremendous therapeutic opportunities in a variety of musculoskeletal diseases. The aims of this study were to generate porous biodegradable scaffolds encapsulating an osteogenic protein, bone morphogenetic protein 2 (BMP-2), and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. BMP-2-encapsulated poly(DL-lactic acid) (PLA) scaffolds were generated by an innovative supercritical fluid process developed for solvent-sensitive and thermolabile growth factors. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on three-dimensional scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor-encapsulated scaffolds was observed after culture in an ex vivo model of bone formation developed on the basis of the chick chorioallantoic membrane model. BMP-2-encapsulated polymer scaffolds showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation and within diffusion chambers implanted into athymic mice as assessed by X-ray analysis and immunocytochemistry. The generation of three-dimensional biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculoskeletal tissue engineering.     

Engineering endochondral bone: in vitro studies

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.     

Histological characterization of the early stages of bone morphogenetic protein-induced osteogenesis

On the basis of currently available knowledge, we hypothesize that the initial bone formation, as induced by bone morphogenetic protein (BMP), is influenced by the chemical composition and three-dimensional spatial configuration of the used carrier material. Therefore, in the current study, the osteoinductive properties of porous titanium (Ti) fiber mesh with a calcium phosphate (Ca-P) coating (Ti-CaP), insoluble bone matrix (IBM), fibrous glass membrane (FGM), and porous particles of hydroxy apatite (PPHAP) loaded with rhBMP-2 were compared in a rat ectopic assay model at short implantation periods. Twelve Ti-CaP, 12 IBM, 12 FGM, and 12 PPHAP implants, loaded with rhBMP-2, were subcutaneously placed in 16 Wistar King rats. The rats were sacrificed at 3, 5, 7, and 9 days post-operative, and the implants were retrieved. Histological analysis demonstrated that IBM and Ti-CaP had induced ectopic cartilage and bone formation by 5 and 7 days, respectively. However, in PPHAP, bone formation and cartilage formation were seen together at 7 days. At 9 days, in Ti-CaP, IBM, and PPHAP, cartilage was seen together with trabecular bone. At 9 days, in FGM, only cartilage was observed. Quantitative rating of the tissue response, using a scoring system, demonstrated that the observed differences were statistically significant (Wilcoxon rank sum test, p < 0.05). We conclude that IBM, CaP-coated Ti mesh, FGM, and PPHAP provided with rhBMP-2 can indeed induce ectopic bone formation with a cartilaginous phase in a rat model at short implantation periods. Considering the different chemical composition and three-dimensional spatial configuration of the carrier materials used, these findings even suggest that endochondral ossification is present in rhBMP-2-induced osteogenesis, even though the amount of cartilage may differ.     

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈86% of the defect area and ≈61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.     

Fate of allogeneic embryonal chick chondrocytes implanted orthotopically, as determined by the host's age

Chondrocytes derived from chick embryos can be successfully implanted in defects of adult chick articular cartilage surfaces. Such implants thrive in their implantation site and create a new articular surface. The chondrocytes mature and hypertrophy in the orthotopic site without invoking an immune response. Eventually a steady state is reached in which mature chondrocytes resurface the defect while in the deeper areas spongy bone replaces the hypertrophic chondrocytes. Time schedules of these repair events have been studied in hosts of different ages. We compared 4-month-old chicks with 3-year-old chickens. The embryonal chondrocytes implanted in the latter group underwent an accelerated aging process. The defects were completely filled-up after 1 month as compared with 2-3 months in the younger age group. Endochondral ossification in the older group was evident as early as 2 months post implantation and was completed after 6 months. This contrasts with the situation in the younger group where the chondrocytes only began to hypertrophy after 6 months. At this stage endochondral ossification was hardly seen at all. A unique response to the cartilaginous implants is seen in the old group only in the vicinity of the reparative tissue, accumulation of hematopoietic centers. This study seem to indicate that the host's environment affects the "biological clock", i.e. rate and degree of aging of the implanted cells, as well as their matrices.     

Development of mature cartilage constructs using novel three-dimensional porous scaffolds for enhanced repair of osteochondral defects

In this study, we successfully developed two types of volume-reduced three-dimensional scaffolds, including cushion- and cylinder-shape scaffolds, fabricated from chitosan-based hyaluronic acid hybrid polymer fibers. Using these scaffolds combined with a bioreactor system, we regenerated histologically and mechanically mature cartilage constructs. The final goal of this study was to clarify the ability of this engineered cartilage construct to induce cartilage repair in osteochondral defects. The mature cartilage constructs regenerated with two types of scaffolds were implanted into 5-mm diameter osteochondral defects in the patellar groove of rabbits. At 12 weeks after implantation, the reparative tissues consisted of hyaline-like cartilage with evidence of stable fusion to adjacent native cartilage and normal reconstitution of subchondral bone. The histological score of these tissues significantly outranked the value of untreated tissue. Biomechanically, compression modulus of reparative tissue at 12 weeks postoperatively was comparative to that of normal articular cartilage. Our results indicate that the implantation of constructs with mature cartilage have potential as a better approach for joint resurfacing.