The use of poly(lactic-co-glycolic acid) microspheres as injectable cell carriers for cartilage regeneration in rabbit knees

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest because it allows cell implantation through minimally invasive surgical procedures. Previously, we showed that poly(lactic-co-glycolic acid) (PLGA) microspheres can be used as an injectable scaffold to engineer cartilage in the subcutaneous space of athymic mice. The purpose of this study was to determine whether PLGA microspheres can be used as an injectable scaffold to regenerate hyaline cartilage in the osteochondral defects of rabbit knees. A full-thickness wound to the patellar groove of the articular cartilage was made in the knees of rabbits. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into these osteochondral wounds. Both chondrocyte transplantations without PLGA microspheres and culture medium injections without chondrocytes served as controls. Sixteen weeks after implantation, chondrocytes implanted using the PLGA microspheres formed white cartilaginous tissues. Histological scores indicating the extent of the cartilaginous tissue repair and the absence of degenerative changes were significantly higher in the experimental group than in the control groups (P < 0.05). Histological analysis by a hematoxylin and eosin stain of the group transplanted with microspheres showed thicker and better-formed cartilage compared to the control groups. Alcian blue staining and Masson's trichrome staining indicated a higher content of the major extracellular matrices of cartilage, sulfated glycosaminoglycans and collagen in the group transplanted with microspheres than in the control groups. In addition, immunohistochemical analysis showed a higher content of collagen type II, the major collagen type in cartilage, in the microsphere transplanted group compared to the control groups. In the group transplanted without microspheres, the wounds were repaired with fibro-cartilaginous tissues. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for cartilage regeneration in a rabbit model of osteochondral wound repair.     

Poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for cartilage tissue engineering

Injectable scaffold has raised great interest for tissue regeneration in vivo, because it allows easy filling of irregularly shaped defects and the implantation of cells through minimally invasive surgical procedures. In this study, we evaluated poly(lactic-co-glycolic acid) (PLGA) microsphere as an injectable scaffold for in vivo cartilage tissue engineering. PLGA microspheres (30-80 microm in diameter) were injectable through various gauges of needles, as the microspheres did not obstruct the needles and microsphere size exclusion was not observed at injection. The culture of chondrocytes on PLGA microspheres in vitro showed that the microspheres were permissive for chondrocyte adhesion to the microsphere surface. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into athymic mouse subcutaneous sites. Chondrocyte transplantation without PLGA microspheres and PLGA microsphere implantation without chondrocytes served as controls. Four and 9 weeks after implantation, chondrocytes implanted with PLGA microspheres formed solid, white cartilaginous tissues, whereas no gross evidence of cartilage tissue formation was noted in the control groups. Histological analysis of the implants by hematoxylin and eosin staining showed mature and well-formed cartilage. Alcian blue/safranin O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosaminoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for in vivo cartilage tissue engineering. This scaffold may be useful to regenerate cartilaginous tissues through minimally invasive surgical procedures in orthopedic, maxillofacial, and urologic applications.     

Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo

The properties of regenerated cartilage using bone marrow-derived mesenchymal stem cells (MSCs) and poly lactic-co-glycolic acid (PLGA) scaffold composites pretreated with TGF-beta3 were investigated and compared to the non-TGF-beta3 treated MSCs/PLGA composites in a rabbit model. We prepared MSCs/PLGA scaffold composites and pretreated it with TGF-beta3 for 3 weeks prior to transplantation. Then, composites were transplanted to the osteochondral defect in the rabbit knee. After 12 weeks of transplantation, 10 of the 12 rabbits in which TGF-beta3 pretreated MSCs/PLGA scaffold composites were transplanted showed cartilaginous regeneration. In gross morphology, regenerated cartilage showed smooth, flush, and transparent features. In indentation test, this had about 80% of Young's modulus of normal articular cartilage. Histological examination demonstrated hyaline like cartilage structures with glycosaminoglycan and type II collagen expression. Histological scores were not statistically different to the normal articular cartilage. These results showed improvement of cartilage regeneration compared to the non-TGF-beta3 pretreated MSCs/PLGA scaffold composite transplanted group. Thus, we have successfully regenerated improved hyaline-like cartilage and determined the feasibility of treating damaged articular cartilage using MSCs/PLGA scaffold composite pretreated with TGF-beta3. Also, we suggest this treatment modality as another concept of cartilage tissue engineering.     

Novel composite hyaluronan/type I collagen/fibrin scaffold enhances repair of osteochondral defect in rabbit knee

A new composite scaffold containing type I collagen, hyaluronan, and fibrin was prepared with and without autologous chondrocytes and implanted into a rabbit femoral trochlea. The biophysical properties of the composite scaffold were similar to native cartilage. The macroscopic, histological, and immunohistochemical analysis of the regenerated tissue from cell-seeded scaffolds was performed 6 weeks after the implantation and predominantly showed formation of hyaline cartilage accompanied by production of glycosaminoglycans and type II collagen with minor fibro-cartilage production. Implanted scaffolds without cells healed predominantly as fibro-cartilage, although glycosaminoglycans and type II collagen, which form hyaline cartilage, were also observed. On the other hand, fibro-cartilage or fibrous tissue or both were only formed in the defects without scaffold. The new composite scaffold containing collagen type I, hyaluronan, and fibrin, seeded with autologous chondrocytes and implanted into rabbit femoral trochlea, was found to be highly effective in cartilage repair after only 6 weeks. The new composite scaffold can therefore enhance cartilage regeneration of osteochondral defects, by the supporting of the hyaline cartilage formation.     

The combined effects of continuous passive motion treatment and acellular PLGA implants on osteochondral regeneration in the rabbit

We investigated the active role of clinical rehabilitation in osteochondral regeneration using continuous passive motion (CPM) treatment together with acellular PLGA implants. CPM treatment was performed and compared with immobilization (Imm) treatment and intermittent active motion (IAM) treatment upon full-thickness osteochondral defects either with or without an PLGA implant in the PI (PLGA-implanted) and ED (empty defect) models. The PI and ED tests were performed in 38 rabbits for 4 and 12 weeks. At the end of testing, the PI-CPM group had the best regeneration with nearly normal articular surfaces and no joint contracture or inflammatory reaction. In contrast, degenerated joints, abrasion cartilage surfaces and synovitis were observed in the Imm and IAM groups. The achieved bone volume/tissue volume (BV/TV) ratio, which was measured using micro-CT, was significantly higher in the CPM group compared with the Imm and IAM groups; in particular, the performance of the PI-CPM group exceeds that of the ED-CPM group. The thickness of the trabecular (subchondral) bone was visibly increased in all of the groups from 4 through 12 weeks of testing. However, a histological analysis revealed differences in cartilage regeneration. At week 4, compared with the ED samples, all of the PI groups exhibited better collagen alignment and higher GAG content in the core of their repaired tissues, particularly in the PI-CPM group. At week 12, sound osteochondral repair and hyaline cartilaginous regeneration was observed in the PI-CPM group, and this was marked by type II collagen expression, osteocyte maturation, and trabecular boney deposition. In contrast, the PI-Imm and PI-IAM groups exhibited fibrocartilaginous tissues that had modest GAG content. In summary, this study demonstrates that early CPM treatment together with acellular PLGA implantation has significant positive effects on osteochondral regeneration in rabbit knee joint models.     

羟基丁酸-羟基辛酸共聚体/胶原骨软骨一体化支架修复膝关节软骨损伤

BACKGROUND: Due to the complex physiological characteristics of the osteochondral tissue, the clinical repair of knee cartilage injury often has dissatisfied outcomes. Tissue engineering methods and tools provide a new idea for osteochondral repair. OBJECTIVE: To observe the effect of poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold on the repair of articular cartilage injury in a rabbit. METHODS: The poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold was prepared by solvent casting/particle leaching method. Then, seed cells were isolated and cultured on the scaffold. Twenty-four healthy New Zealand white rabbits, 4 weeks of age, were used for the study. Under balanced anesthesia, an articular cartilage defect (4.5 mm in diameter, 5 mm in depth) was created on the rabbit’s femoral condyle using a bone drill. After modeling, rabbits were randomized into three groups and given direct suture in blank group, pure scaffold implantation in control group and implantation of the scaffold-cell complex in experimental group. Femoral condyle of each rabbit was taken out for gross and histological observations at 8, 20 weeks after surgery. RESULTS AND CONCLUSION: At 8 weeks after surgery, transparent film-covered defects and small/irregular cells were found in the experimental group; the defects were filled with fibrous tissues in the control group; while there was no repair in the blank group. Until the 20th week, the defects were covered with hyaline cartilage-like tissues, accompanied by regular cell arrangement in the experimental group; in the control group, the defects were covered with white membranous tissues, and many chondrocytes were found at the basement and edge; in the blank group, some newborn tissues were visible at the defect region. These findings suggest that the poly (hydroxybutyrate-co- hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold carrying seed cells contributes to articular cartilage repair.     

Use of a biphasic graft constructed with chondrocytes overlying a beta-tricalcium phosphate block in the treatment of rabbit osteochondral defects

To evaluate the ability of a biphasic construct to repair osteochondral defects in articular cartilage, plugs made of chondrocytes in collagen gel overlying a resorbable porous beta-tricalcium phosphate (TCP) block were implanted into defects in rabbit knees. The repair tissue was evaluated at 8, 12, and 30 weeks. Eight weeks after implantation of the biphasic construct, histologic examination showed hyaline-like cartilage formation that was positive for safranin O and type II collagen. At 12 weeks, most of the beta-TCP was replaced by bone, with a small amount remaining in the underlying cartilage. In the cell-seeded layer, the newly formed middle and deep cartilage adjacent to the subchondral bone stained with safranin O, but no staining was observed in the superficial layer. In addition, cell morphology was distinctly different from the deep levels of the reparative cartilage, with hypertrophic cells at the bottom of the cartilaginous layer. At 30 weeks, beta-TCP had completely resorbed and a tidemark was observed in some areas. In contrast, controls (defects filled with a beta-TCP block alone) showed no cartilage formation but instead had subchondral bone formation. These findings indicate that beta-TCP-supported chondrocytes in collagen gel can partially repair isolated articular cartilage osteochondral defects.     

Autologous chondrocyte implantation with collagen bioscaffold for the treatment of osteochondral defects in rabbits

Osteochondral injury is therapeutically irreversible within current treatment parameters. Autologous chondrocyte implantation (ACI) promises to regenerate hyaline articular cartilage, but conventional ACI is plagued by complications determined by periosteal grafting. Here we propose the utilization of collagen membrane in ACI as an effective bioscaffold for the regeneration of osteochondral lesions. Using a rabbit model of osteochondral injury, we have inoculated autologous chondrocytes onto a type I/III collagen scaffold [so-called matrix-induced ACI (MACI)] and implanted into 3-mm osteochondral knee defects. All untreated defect histology showed inferior fibrocartilage and/or fibrous tissue repair. In our time-course study, ACI with type I/III collagen membrane regenerated cartilage with healthy osteochondral architecture in osteochondral defects at 6 weeks. At 12 weeks, articular cartilage regeneration was maintained, with reduced thickness and proteoglycan compared with the adjacent cartilage. Both 6-week (p < 0.01) and 12-week (p < 0.05) ACI with collagen membrane showed significant improvement as compared with untreated controls. To further examine the efficacy of cartilage regeneration by ACI, we conducted a dose-response study, using chondrocytes at various cell densities between 10(4) and 10(6) cells/cm(2). The results showed that cell density had no effect on outcome histology, but all cell densities were significantly better than untreated controls (p < 0.01) and cell-free collagen membrane treatment (p < 0.05). In short, our data suggest that autologous chondrocyte-seeded type I/III collagen membrane is an effective method for the treatment of focal osteochondral knee injury in rabbits.     

同种异体和异种来源骨髓间充质干细胞诱导成软骨修复喉软骨缺损

BACKGROUND: Because chondrocytes have no regeneration ability, to select suitable seed cells is the primary problem to repair cartilage defects. OBJECTIVE: To investigate the effect of allogeneic versus heterologous bone marrow mesenchymal stem cells (BMSCs) in repairing laryngeal cartilage defects after chondrogenic induction. METHODS: BMSCs from human and rabbits were isolated and cultured. Passage 3 cells were cultured in chondrogenic induction medium containing transforming transforming growth factor beta 1 and bone morphogenetic protein, and then were dropped onto a poly(lactic-co-glycolic acid) (PLGA) scaffold. Thirty New Zealand rabbits were randomly assigned into three groups: blank control group, human BMSCs group, rabbit BMSCs group. Animal models of laryngeal cartilage defects were made in the three groups. After modeling, saline-soaked PLGA scaffold, PLAG scaffold with human BMSCs or with rabbit BMSCs were implanted respectively into the rabbits in the normal blank, human BMSCs and rabbit BMSCs groups. The expression of type II collagen in the larynx and its surrounding tissues was detected by immunohistochemistry at 4 and 8 weeks postoperatively. RESULTS AND CONCLUSION: The animals in each group breathed normally with no presence of wheezing, and their eating and activity were good. Moreover, there was no purulency or infection in the three groups. At 4 and 8 weeks after operation, the positive rates of type II collagen in the two BMSCs groups were significantly higher than that in the blank control group (P < 0.05). There was no significant difference between two BMSCs groups (P > 0.05). These results show that both allogeneic and heterologous BMSCs have good therapeutic effects on the repair of laryngeal cartilage defects in rabbits.      

Apatite-coated poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for bone tissue engineering

Biodegradable polymer/ceramic composite scaffold could overcome limitations of biodegradable polymers or ceramics for bone regeneration. Injectable scaffold has raised great interest for bone regeneration in vivo, since it allows one for easy filling of irregularly shaped bone defects and implantation of osteogenic cells through minimally invasive surgical procedures The purpose of this study was to determine whether apatite-coated poly(lactic-co-glycolic acid) (PLGA) microspheres could be used as an injectable scaffold to regenerate bone in vivo. Apatite-coated PLGA microspheres were fabricated by incubating PLGA microspheres in simulated body fluid. The apatite that coated the PLGA microsphere surfaces was similar to apatite in natural bone, as demonstrated by scanning electron microscopy, X-ray diffraction spectra, energy-dispersive spectroscopy, and Fourier transformed-infrared spectroscopy analyses. Rat osteoblasts were mixed with apatite-coated PLGA microspheres and injected immediately into subcutaneous sites of athymic mice. Osteoblast transplantation with plain PLGA microspheres served as a control. Histological analysis of the implants at 6 weeks with hematoxylin and eosin staining, Masson's trichrome staining, and von Kossa staining revealed much better regeneration of bone in the apatite-coated PLGA microsphere group than the plain PLGA microsphere group. The new bone formation area and the calcium content of the implants were significantly higher in the apatite-coated PLGA microsphere group than in the plain PLGA microsphere group. This study demonstrates the feasibility of using apatite-coated PLGA microspheres as an injectable scaffold for in vivo bone tissue engineering. This scaffold may be useful for bone regeneration through minimally invasive surgical procedures in orthopedic applications.     

The Effect of Osteochondral Regeneration Using Polymer Constructs and Continuous Passive Motion Therapy in the Lower Weight-Bearing Zone of Femoral Trocheal Groove in Rabbits

Remedying patellofemoral osteochondral defects using clinical therapy remains challenging. Construct-based and cell-based regenerative medicine with in vitro physical stimuli has been progressively implemented. However, the effect of physical stimuli in situ in knee joints with degradable constructs is still not well-documented. Therefore, we studied whether it was practical to achieve articular cartilage repair using a poly(lactic-co-glycolic acid) (PLGA) construct in addition to early short-term continuous passive motion (CPM) for treatment of full-thickness osteochondral defects in the lower-weigh bearing (LWB) zone of the femoral trocheal groove. Twenty-six rabbits were randomly allocated into either intermittent active motion (IAM) or CPM treatment groups with or without PLGA constructs, termed PLGA construct-implanted (PCI) and empty defect knee models, respectively. Gross observation, histology, inflammatory cells, which were identified using H&E staining, total collagen and alignment, studied qualitatively using Masson’s trichrome staining, glycosaminoglycan (GAG), identified using Alcian blue staining, and newly formed bone, observed using micro-CT, were evaluated at 4 and 12 weeks after surgery. Repair of osteochondral defects in the PCI-CPM group was more promising than all other groups. The better osteochondral defect repair in the PCI-CPM group corresponded to smooth cartilage surfaces, no inflammatory reaction, hyaline cartilaginous tissues composition, sound collagen alignment with positive collagen type II expression, higher GAG content, mature bone regeneration with osteocyte, clear tidemark formation, and better degradation of PLGA. In summary, the use of a simple PLGA construct coupled with passive motion promotes positive healing and may be a promising clinical intervention for osteochondral regeneration in LWB defects.     

Cartilage regeneration with highly-elastic three-dimensional scaffolds prepared from biodegradable poly(L-lactide-co-epsilon-caprolactone)

Compressive mechanical stimuli are crucial in regenerating cartilage with tissue engineering, which creates a need for scaffolds that can maintain their mechanical integrity while delivering mechanical signals to adherent cells during strain applications. With these goals in mind, the aim of this study was to develop a mechano-active scaffold that facilitated effective cartilaginous tissue formation under dynamic physiological environments. Using a gel-pressing method, we fabricated a biodegradable and highly-elastic scaffold from poly(L-lactide-co-epsilon-caprolactone) (PLCL; 5:5), with 85% porosity and a 300-500-microm pore size, and we compared it to control scaffolds made of rigid polylactide (PLA) or poly(lactide-co-glycolide) (PLGA). After tensile mechanical tests and recovery tests confirmed the elasticity of the PLCL scaffolds, we seeded them with rabbit chondrocytes, cultured them in vitro, and subcutaneously implanted them into nude mice for up to eight weeks. The PLCL scaffolds possessed a completely rubber-like elasticity, were easily twisted and bent, and exhibited an almost complete (over 97%) recovery from applied strain (up to 500%); the control PLA scaffolds showed little recovery. In vitro and in vivo accumulations of extracellular matrix on the cell-PLCL constructs demonstrated that they could not only sustain but also significantly enhance chondrogenic differentiation. Moreover, the mechanical stimulation of the dynamic in vivo environment promoted deposition of the chondral extracellular matrix onto the PLCL. In contrast, on the PLA scaffolds, most of the chondrocytes had de-differentiated and formed fibrous tissues. In a rabbit defect model, the groups treated with PLCL scaffolds exhibited significantly enhanced cartilage regeneration compared to groups harboring an empty control or PLGA scaffolds. These results indicated that the mechano-active PLCL scaffolds effectively delivered mechanical signals associated with biological environments to adherent chondrocytes, suggesting that these elastic PLCL scaffolds could successfully be used for cartilage regeneration.     

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.     

The use of de-differentiated chondrocytes delivered by a heparin-based hydrogel to regenerate cartilage in partial-thickness defects

Partial-thickness cartilage defects, with no subchondral bone injury, do not repair spontaneously, thus there is no clinically effective treatment for these lesions. Although the autologous chondrocyte transplantation (ACT) is one of the promising approaches for cartilage repair, it requires in vitro cell expansion to get sufficient cells, but chondrocytes lose their chondrogenic phenotype during expansion by monolayer culture, leading to de-differentiation. In this study, a heparin-based hydrogel was evaluated and optimized to induce cartilage regeneration with de-differentiated chondrocytes. First, re-differentiation of de-differentiated chondrocytes encapsulated in heparin-based hydrogels was characterized in vitro with various polymer concentrations (from 3 to 20 wt.%). Even under a normal cell culture condition (no growth factors or chondrogenic components), efficient re-differentiation of cells was observed with the optimum at 10 wt.% hydrogel, showing the complete re-differentiation within a week. Efficient re-differentiation and cartilage formation of de-differentiated cell/hydrogel construct were also confirmed in vivo by subcutaneous implantation on the back of nude mice. Finally, excellent cartilage regeneration and good integration with surrounding, similar to natural cartilage, was also observed by delivering de-differentiated chondrocytes using the heparin-based hydrogel in partial-thickness defects of rabbit knees whereas no healing was observed for the control defects. These results demonstrate that the heparin-based hydrogel is very efficient for re-differentiation of expanded chondrocytes and cartilage regeneration without using any exogenous inducing factors, thus it could serve as an injectable cell-carrier and scaffold for cartilage repair. Excellent chondrogenic nature of the heparin-based hydrogel might be associated with the hydrogel characteristic that can secure endogenous growth factors secreted from chondrocytes, which then can promote the chondrogenesis, as suggested by the detection of TGF-β1 in both in vitro and in vivo cell/hydrogel constructs.     

Bioactive Stratified Polymer Ceramic-Hydrogel Scaffold for Integrative Osteochondral Repair

Due to the intrinsically poor repair potential of articular cartilage, injuries to this soft tissue do not heal and require clinical intervention. Tissue engineered osteochondral grafts offer a promising alternative for cartilage repair. The functionality and integration potential of these grafts can be further improved by the regeneration of a stable calcified cartilage interface. This study focuses on the design and optimization of a stratified osteochondral graft with biomimetic multi-tissue regions, including a pre-designed and pre-integrated interface region. Specifically, the scaffold based on agarose hydrogel and composite microspheres of polylactide-co-glycolide (PLGA) and 45S5 bioactive glass (BG) was fabricated and optimized for chondrocyte density and microsphere composition. It was observed that the stratified scaffold supported the region-specific co-culture of chondrocytes and osteoblasts which can lead to the production of three distinct yet continuous regions of cartilage, calcified cartilage and bone-like matrices. Moreover, higher cell density enhanced chondrogenesis and improved graft mechanical property over time. The PLGA-BG phase promoted chondrocyte mineralization potential and is required for the formation of a calcified interface and bone regions on the osteochondral graft. These results demonstrate the potential of the stratified scaffold for integrative cartilage repair and future studies will focus on scaffold optimization and in vivo evaluations.     

Radially oriented collagen scaffold with SDF-1 promotes osteochondral repair by facilitating cell homing

The migration of cells from the side and the bottom of the defect is important in osteochondral defect healing. Here, we designed a novel collagen scaffold that possessed channels in both the horizontal and the vertical directions, along with stromal cell-derived factor-1 (SDF-1) to enhance osteochondral regeneration by facilitating cell homing. Firstly we fabricated the radially oriented and random collagen scaffolds, then tested their properties. The radially oriented collagen scaffold had better mechanical properties than the random scaffold, but both supported cell proliferation well. Then we measured the migration of BMSCs in the scaffolds in vitro. The radially oriented collagen scaffold effectively promoted their migration, and this effect was further facilitated by addition of SDF-1. Moreover, we created osteochondral defects in rabbits, and implanted them with random or oriented collagen scaffolds with or without SDF-1, and evaluated cartilage and subchondral bone regeneration at 6 and 12 weeks after surgery. Cartilage regeneration with both the radially oriented scaffold and SDF-1 effectively promoted repair of the cartilage defect. Our results confirmed that the implantation of the radially oriented channel collagen scaffold with SDF-1 could be a promising strategy for osteochondral repair.     

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells–fibrin suspension and polymerized by dropping thrombin–sodium chloride (CaCl₂) solution. A PLGA–cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold–cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.     

Cell-based tissue-engineered allogeneic implant for cartilage repair

The potential for using of allogeneic cartilage chips, transplanted in a biologic polymer with articular chondrocytes, as a tool for articular cartilage repair was studied. Small lyophilized articular cartilage chips were mixed with a cell/fibrinogen solution and thrombin to obtain implantable constructs made of fibrin glue, chondrocytes, and cartilage chips. Specimens were implanted in the subcutaneous tissue on the backs of nude mice (experimental group A). Three groups of controls (groups B, C, and D) were also prepared. Group B consisted of fibrin glue and cartilage chips without chondrocytes. Group C consisted of fibrin glue and chondrocytes without cartilage chips, and group D was composed solely of fibrin glue. All samples were carefully weighed before implantation in the mice. The constructs were harvested from the animals at 6, 9, and 12 weeks, examined grossly, and weighed. The samples were then processed and stained with hematoxylin and eosin for histological examination. Gross evaluation and weight analysis of the constructs at the time of retrieval showed retention of the original mass in the samples made of fibrin glue, chondrocytes, and cartilage chips (group A) and demonstrated a cartilaginous consistency upon probing. Specimens from constructs of fibrin glue and cartilage chips without chondrocytes (control group B) retained most of their volume, but were statistically lighter than specimens from group A and were much softer and more pliable than those in group A. Samples of specimens from constructs of fibrin glue and chondrocytes (groups C) and fibrin glue alone (group D) both showed a substantial reduction of their original masses over the experimental time periods when compared to the samples in groups A and B, although specimens from group C demonstrated new cartilage matrix formation. Histological analysis of specimens in experimental group A demonstrated the presence of cartilage chips surrounded by newly formed cartilaginous matrix, while specimens of control group B showed only fibrotic tissue surrounding the devitalized cartilage pieces. Cartilaginous matrix was also observed in control group C, in which cartilage chips were absent, whereas only fibrin glue debris was observed in control group D. This study demonstrated that a composite of fibrin glue and devitalized cartilage can serve as a scaffold for chondrocyte transplantation, preserve the original phenotype of the chondrocytes, and maintain the original mass of the implant. This may represent a valid option for addressing the problem of articular cartilage repair.     

A Collagen–Poly(Vinyl Alcohol) Nanofiber Scaffold for Cartilage Repair

Articular cartilage has a limited capacity for self-repair. Untreated injuries of cartilage may lead to osteoarthritis. This problem demands new effective methods to reconstruct articular cartilage. Mesenchymal stem cells (MSCs) have the proclivity to differentiate along multiple lineages giving rise to new bone, cartilage, muscle, or fat. This study was an animal model for autologous effects of transplantation of MSCs with a collagen–poly(vinyl alcohol) (PVA) scaffold into full-thickness osteochondral defects of the stifle joint in the rabbit as an animal model. A group of 10 rabbits had a defect created experimentally in the full thickness of articular cartilage penetrated into the subchondral space in the both stifle joints. The defect in the right stifle was filled with MSCs/collagen–PVA scaffold (group I), and in the left stifle, the defect was left without any treatment as the control group (group II). Specimens were harvested at 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type-II collagen. Histology observation showed that the MSCs/collagen–PVA repair group had better chondrocyte morphology, continuous subchondral bone, and much thicker newly formed cartilage compared with the control group at 12 weeks post operation. There was a significant difference in histological grading score between these two groups. The present study suggested that the hybrid collagen–PVA scaffold might serve as a new way to keep the differentiation of MSCs for enhancing cartilage repair.     

Evaluation of three-dimensional scaffolds prepared from poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for growth of allogeneic chondrocytes for cartilage repair in rabbits

Articular cartilage repair using tissue engineering approach generally requires the use of an appropriate scaffold architecture that can support the formation of cartilage tissue. In this investigation, the potential of three-dimensional scaffolds made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated in rabbit articular cartilage defect model. Engineered PHBHHx cartilage constructs inoculated in vitro with rabbit chondrocytes for 30 days were examined. Subsequently the constructs inoculated with chondrocytes for 10 days were selected for transplantation into rabbits. After 16 weeks of in vivo implantation, both the engineered cartilage constructs and the bare scaffolds were found to be filled the defects with white cartilaginous tissue, with the engineered constructs showing histologically good subchondral bone connection and better surrounding cartilage infusion. Owing to pre-seeded chondrocytes in the PHBHHx scaffolds, better surface integrality and more accumulation of extracellular matrix (ECM) including type II collagen and sGAG were achieved in the engineered cartilage constructs. The repaired tissues possessed an average compressive modulus of 1.58MPa. For comparison, the defects without repair treatments still showed defects with fibrous tissues. These results demonstrated that PHBHHx is a useful material for cartilage tissue engineering.