Articular cartilage reconstruction using xenogeneic epiphyses slices

Reconstruction of articular cartilage defects using adult osteochondral allografts is an established clinical procedure, whose principal drawback is lack of lateral integration of the grafts to the surrounding tissue. Autologous chondrocytes transplantation is a sophisticated technique requiring cell culture and a staged operation. Its main draw back is the lack of mechanical strength early on. This study was conducted in order to evaluate the possibility of using embryonal epiphyses as a cartilage reconstruction tissue. A xenogeneic human to rabbit sub-acute osteochondral defect model was designed to evaluate the possibility of allogeneic implantation in humans. The following procedures were perfomed (n = 5): transplantation of 1. live epiphyses 2. live epiphyses with autogeneic periosteum 3. de-vitalized epiphyses and 4. devitalized epiphyses with autogeneic articular chondrocytes. A fifth control group did not receive any implant. Animals in groups 1 and 2 had a viable reconstruction of the articular surface with little evidence of rejection and without pannus formation. Animals in groups 3 and 4 became severely arthritic and the graft was resorbed. Nitric oxide synthase accumulation was reduced in group 1 and 2 as compared to groups 3, 4, and 5, indicating a joint preserving function of the epiphyseal grafts. Epiphyseal grafts appear to be a feasible procedure for reconstruction of articular cartilage defects even in a xenogeneic model. This revised version was published online in July 2006 with corrections to the Cover Date.     

A semiquantitative scale for histologic grading of articular cartilage repair.

This laboratory has developed a semiquantitative scale for grading the natural healing process of defects drilled into articular cartilage. The scale is composed of four parameters: percent filling of the defect, reconstitution of the osteochondral junction, matrix staining and cell morphology; it has a score range from 0 (best) to 14 (worst). The scale was used to evaluate the healing of defects drilled into rabbit knee articular cartilage at 2, 14, 30, 60 and 120 days after surgery. No statistically significant difference in the graded score was found between the two different defect sizes (2.7 and 1.5 mm). However, the differences in score observed between specimens from different sacrifice times were significant (p less than 0.01). Currently many investigators are manipulating cartilaginous lesions in an attempt to improve healing, and this scale will provide a means for quantitatively comparing results from control and experimental groups.     

Influence of freezing with liquid nitrogen on whole-knee joint grafts and protection of cartilage from cryoinjury in rabbits

Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24 h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.     

Long-term clinical experience with fresh osteochondral allografts for articular knee defects in high demand patients

Fresh osteochondral allografts are used to repair osteoarticular defects of the knee. For post-traumatic defects recent advances in other techniques for cartilage repair and resurfacing have reduced the role of allograft tissue transplantation to defects larger than 3 cm in diameter and 1 cm in depth. A fresh osteochondral allograft that has been harvested from a donor within 24 h from death and preserved in 4°C for up to 4 days shows 100% viability of the cartilage. The avascular bone remains structurally intact and mechanically strong until it is replaced by host bone or until it is weakened or absorbed. The indications for fresh osteochondral allografts for reconstructive surgery of the articular surface of the knee do not justify the use of immunosuppressive drugs and we therefore believe that surgical vascularization of the grafts should not be carried out. This clinical approach can provide a reconstructive solution for younger higher demand patients where implants are not desirable and arthrodesis is not acceptable. A clinical follow-up study as early as 1975 showed successful early outcomes. More recently, survival analysis found 95% survival at 5 years, 71% at 10 years, and 66% at 20 years. It was learned that older patients, bipolar transplants, improper loading of the graft, and grafts for osteoarthritis and steroid-induced avascular necrosis do not lead to good long-term outcomes. We would like to describe here some of our long-term clinical experience concerning this surgery. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transplantation of autologous rabbit BM-derived mesenchymal stromal cells embedded in hyaluronic acid gel sponge into osteochondral defects of the knee

BACKGROUND: Mesenchymal stromal cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, suggesting that these cells are an attractive cell source for cartilage tissue engineering approaches. Various methods, such as using hyaluronan-based materials, have been employed to improve transplantation for repair. Our objective was to study the effects of autologous transplantation of rabbit MSC with hyaluronic acid gel sponges into full-thickness osteochondral defects of the knee. METHODS: Rabbit BM-derived MSC were cultured and expanded with fibroblast growth factor (FGF). Specimens were harvested at 4 and 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type II collagen and CD44. RESULTS: The regenerated area after autologous transplantation of hyaluronic acid gel sponge loaded with MSC into the osteochondral defect at 12 weeks after surgery showed well-repaired cartilage tissue, resembling the articular cartilage of the surrounding structure, of which the histologic score was significantly better than that of the untreated osteochondral defect. In the regenerated cartilage, type II collagen was found in the pericellular matrix of regenerative chondrocytes, while CD44 expression in the regenerative tissue could not be revealed. DISCUSSION: These data suggest that the autologous transplantation of MSC embedded in hyaluronan-based material may support chondrogenic differentiation and be useful for osteochondral defect repair.     

Development of a preclinical natural porcine knee simulation model for the tribological assessment of osteochondral grafts in vitro

In order to pre-clinically evaluate the performance and efficacy of novel osteochondral interventions, physiological and clinically relevant whole joint simulation models, capable of reproducing the complex loading and motions experienced in the natural knee environment are required. The aim of this study was to develop a method for the assessment of tribological performance of osteochondral grafts within an in vitro whole natural joint simulation model.The study assessed the effects of osteochondral allograft implantation (existing surgical intervention for the repair of osteochondral defects) on the wear, deformation and damage of the opposing articular surfaces. Tribological performance of osteochondral grafts was compared to the natural joint (negative control), an injury model (focal cartilage defects) and stainless steel pins (positive controls). A recently developed method using an optical profiler (Alicona Infinite Focus G5, Alicona Imaging GmbH, Austria) was used to quantify and characterise the wear, deformation and damage occurring on the opposing articular surfaces. Allografts inserted flush with the cartilage surface had the lowest levels of wear, deformation and damage following the 2 h test; increased levels of wear, deformation and damage were observed when allografts and stainless steel pins were inserted proud of the articular surface. The method developed will be applied in future studies to assess the tribological performance of novel early stage osteochondral interventions prior to in vivo studies, investigate variation in surgical precision and aid in the development of stratified interventions for the patient population.     

Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Cartilage graft is considered to be useful in repairing chondral or osteochondral defects. One method of the cartilage graft is achieved by autologous chondrocyte transplantation following cell culture. However, chondrocytes change their phenotype during culture. We used costal chondrocytes cultured over agarose (suspension culture) as a source of graft materials. The suspension-cultured chondrocytes formed aggregate in culture. We first examined the expressions of cartilage-specific matrices of cultured chondrocytes after two weeks in culture. The chondrocytes cultured over agarose expressed more type II collagen mRNA than those cultured on plastic dishes did after two weeks in culture. Safranin O staining showed the presence of glycosaminoglycans in the chondrocyte culture over agarose, while glycosaminoglycans were not observed in the culture on plastic dishes. We then examined the changes of rat articular osteochondral defects after transplantation of suspension-cultured chondrocytes. The aggregate of suspension-cultured chondrocytes was easily picked up with forceps and transplanted in the osteochondral defects. The defects were filled with safranin O-stained hyaline cartilage tissue two weeks after chondrocyte transplantation. On the contrary, the fibrous materials, which were not stained with safranin O, were observed in the control defects. These results suggest that the suspension-cultured chondrocytes are useful for autologous cartilage grafts by preserving chondrocyte phenotype.     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

Periarticular cancellous bone changes following anterior cruciate ligament injury.

To understand more fully the early bone changes in an experimental model of osteoarthrosis, we quantified periarticular bone mineral density and bone mechanical properties in anterior cruciate ligament transected (ACLX) knee joints (4, 10, 32, and 39 wk post-ACLX) compared with contralateral joints and unoperated normal joints of skeletally mature animals. Maximal stress and energy were significantly reduced in ACLX cancellous bone from the medial femoral condyles at 4 wk postinjury. All mechanical properties (e.g., yield stress and elastic modulus) declined after 4 wk and were significantly reduced at 10 wk. ACLX bone mineral density was significantly reduced at all measured time points. Ash content was significantly reduced at 10 and 32 wk. Changes in the lateral condyles were similar but less pronounced than in the medial condyles. These bony changes accompanied the earliest articular cartilage molecular changes and preceded changes in the articular cartilage gross morphology. We suggest that these early changes in bone mechanical behavior contribute to the progression of osteoarthrosis and pathogenic changes in the joint.     

Reliability of cartilage digestion and FDA–EB fluorescence staining for the detection of chondrocyte viability in osteochondral grafts

The purpose of this study is to evaluate the reliability of cartilage digestion and fluorescein diacetate-ethidium bromide (FDA–EB) fluorescence staining for the detection of chondrocyte viability in osteochondral grafts. Sixteen fresh osteochondral grafts were harvested from pig knee condyles, and the articular cartilage tissue was preserved. Each cartilage graft was cut into two 70-µm thick pieces and randomly allocated to Group A or Group B. The cell viability of Group A was detected using FDA–EB fluorescence staining of the digested cartilage, and the viability of Group B was detected with FDA–EB fluorescence staining of cartilage sections. Comparisons of chondrocyte viability and correlation analyses of the two groups were performed using the paired sample t test and Pearson correlation test, respectively. No significant difference was found in the chondrocyte viability between Groups A and B (p > 0.05), and a strong correlation was observed (r = 0.70, p < 0.05). Therefore, cartilage digestion with FDA–EB fluorescence staining is a reliable method for detecting chondrocyte viability in osteochondral grafts.     

Experimental studies on repair of large osteochondral defects at a high weight bearing area of the knee joint: a tissue engineering study

There is a vast clinical need for the development of an animal model to study the fundamentals of healing of injured or diseased diarthrodial joints (knee, hip, shoulder, wrist, etc). Current prosthetic replacements do not offer acceptable treatment for injuries and diseases of these joints in young active individuals. New clinical treatment modalities, based on sound biologic principles, are sought for the development of repair or healing tissues engineered to have similar biomechanical properties as normal articular cartilage. In this paper we present a brief review of this need, and propose a grafting procedure which may lead to a successful animal model for studies of long term repair of major osteochondral defects. This grafting procedure uses an autologous periosteum-bone graft or an autologous-synthetic bone replacement graft. We have applied these grafts for in vivo repair of large surgically created defects in the high weight bearing area of the distal femoral condyle of mature New Zealand white rabbits. Further, an interdisciplinary study, including histochemistry, biochemistry (composition and metabolic activities), and biomechanics (biphasic properties), was performed to assess the feasibility of our animal model to generate viable repair tissues. We found our grafting procedure produced, 8 weeks postoperatively, tissues which were very similar to those found in normal articular cartilage. However, our histological studies indicate incomplete bonding between the repair tissue and the adjacent cartilage, and lack of an appropriate superficial zone at the articular surface. These deficiencies may cause long term failure of the repair tissue. Further studies must be undertaken to enhance development of a strong bond and a collagen-rich surface zone. This may require the use of growth factors (e.g., transforming growth factors beta) capable of simulating extra collagen production, or the use of serum derived tissue glue for bonding. At present, we are pursuing these studies.     

The minipig model for experimental chondral and osteochondral defect repair in tissue engineering: retrospective analysis of 180 defects

Articular cartilage repair is still a challenge in orthopaedic surgery. Although many treatment options have been developed in the last decade, true regeneration of hyaline articular cartilage is yet to be accomplished. In vitro experiments are useful for evaluating cell-matrix interactions under controlled parameters. When introducing new treatment options into clinical routine, adequate animal models are capable of closing the gap between in vitro experiments and the clinical use in human beings. We developed an animal model in the G?ttingen minipig (GMP) to evaluate the healing of osteochondral or full-thickness cartilage defects. The defects were located in the middle third of the medial portion of the patellofemoral joint at both distal femurs. Chondral defects were 6.3 mm, osteochondral defects either 5.4 or 6.3 mm in diameter and 8 or 10 mm deep. In both defects the endogenous repair response showed incomplete repair tissue formation up to 12 months postoperatively. Based on its limited capability for endogenous repair of chondral and osteochondral defects, the GMP is a useful model for critical assessment of new treatment strategies in articular cartilage tissue engineering.     

Transforming growth factor beta superfamily members: role in cartilage modeling

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover.     

The effect of storage on the biomechanical behavior of articular cartilage--a large strain study.

The transplantation of stored shell osteochondral allografts is a potentially useful alternative to total joint replacements for the treatment of joint ailments. The maintenance of normal cartilage properties of the osteochondral allografts during storage is important for the allograft to function properly and survive in the host joint. Since articular cartilage is normally under large physiological stresses, this study was conducted to investigate the biomechanical behavior under large strain conditions of cartilage tissue stored for various time periods (i.e., 3, 7, 28, and 60 days) in tissue culture media. A biphasic large strain theory developed for soft hydrated connective tissues was used to describe and determine the biomechanical properties of the stored cartilage. It was found that articular cartilage stored for up to 60 days maintained the ability to sustain large compressive strains of up to 40 percent or more, like normal articular cartilage. Moreover, the equilibrium stress-strain behavior and compressive modulus of the stored articular cartilage were unchanged after up to 60 days of storage.     

Mechano-regulation of stem cell differentiation and tissue regeneration in osteochondral defects

Cartilage defects that penetrate the subchondral bone can undergo spontaneous repair through the formation of a fibrous or cartilaginous tissue mediated primarily by mesenchymal stem cells from the bone marrow. This tissue is biomechanically inferior to normal articular cartilage, and is often observed to degrade over time. Whether or not biomechanical factors control the type and quality of the repair tissue, and its subsequent degradation, have yet to be elucidated. In this paper, we hypothesise a relationship between the mechanical environment of mesenchymal stem cells and their subsequent dispersal, proliferation, differentiation and death. The mechano-regulation stimulus is hypothesised to be a function of strain and fluid flow; these quantities are calculated using biphasic poroelastic finite element analysis. A finite element model of an osteochondral defect in the knee was created, and used to simulate the spontaneous repair process. The model predicts bone formation through both endochondral and direct intramembranous ossification in the base of the defect, cartilage formation in the centre of the defect and fibrous tissue formation superficially. Greater amounts of fibrous tissue formation are predicted as the size of the defect is increased. Large strains are predicted within the fibrous tissue at the articular surface, resulting in significant cell apoptosis. This result leads to the conclusion that repair tissue degradation is initiated in the fibrous tissue that forms at the articular surface. The success of the mechano-regulation model in predicting many of the cellular events that occur during osteochondral defect healing suggest that in the future it could be used as a tool for optimising scaffolds for tissue engineering.     

Protein kinase C delta null mice exhibit structural alterations in articular surface,intra-articular and subchondral compartments

IntroductionStructural alterations in intra-articular and subchondral compartments are hallmarks of osteoarthritis, a degenerative disease that causes pain and disability in the aging population. Protein kinase C delta (PKC-δ) plays versatile functions in cell growth and differentiation, but its role in the articular cartilage and subchondral bone is not known.MethodsHistological analysis including alcian blue, safranin O staining and fluorochrome labeling were used to reveal structural alterations at the articular cartilage surface and bone–cartilage interface in PKC-δ knockout (KO) mice. The morphology and organization of chondrocytes were studied using confocal microscopy. Glycosaminoglycan content was studied by micromass culture of chondrocytes of PKC-δ KO mice.ResultsWe uncovered atypical structural demarcation between articular cartilage and subchondral bone of PKC-δ KO mice. Histology analyses revealed a thickening of the articular cartilage and calcified bone–cartilage interface, and decreased safranin O staining accompanied by an increase in the number of hypertrophic chondrocytes in the articular cartilage of PKC-δ KO mice. Interestingly, loss of demarcation between articular cartilage and bone was concomitant with irregular chondrocyte morphology and arrangement. Consistently, in vivo calcein labeling assay showed an increased intensity of calcein labeling in the interface of the growth plate and metaphysis in PKC-δ KO mice. Furthermore, in vitro culture of chondrocyte micromass showed a decreased alcian blue staining of chondrocyte micromass in the PKC-δ KO mice, indicative of a reduced level of glycosaminoglycan production.ConclusionsOur data imply a role for PKC-δ in the osteochondral plasticity of the interface between articular cartilage and the osteochondral junction.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0720-4) contains supplementary material, which is available to authorized users.     

Effect of isolation of periosteum and dura on the healing of rabbit calvarial inlay bone grafts

Little is understood about the role of the recipient site in the revascularization and incorporation of autogenous inlay bone grafts in the craniofacial skeleton. Clinical experience demonstrates that secondary complex cranial vault reconstruction performed with scarred avascular dura or poor soft-tissue coverage may undergo significant resorption, thus compromising the aesthetic outcome. This study was designed to determine the effect of isolating autogenous orthotopic inlay calvarial bone grafts from the surrounding dura and/or periosteum on graft revascularization, healing, and volume maintenance in the adult rabbit. Adult rabbits were randomized into four groups (n = 10 per group); in each rabbit, the authors created a circular, 15-mm in diameter, full-thickness cranial defect followed by reconstruction with an autogenous calvarial bone graft, which was replaced orthotopically and held with microplate fixation. Silicone sheeting (0.5 mm thickness) was used to isolate the dura (group II), the periosteum (group II), or both dura and periosteum (group IV) from the graft interface. No silicone was placed in group I. Animals were killed 10 weeks postoperatively, and calvaria were harvested to assess graft surface area, morphology, quantitative histology, fluorochrome staining, and revascularization. Grafts isolated from both the dura and periosteum exhibited significant decreases in total bone (cortical and trabecular) surface area, blood vessel count, and interface healing compared with nonisolated control grafts. Isolation of either the dura or periosteum significantly (p < 0.05) decreased blood vessel count but had no significant effect on interface healing. Isolation of the dura alone was associated with a significant (p < 0.05) decrease in graft cross-sectional surface area and dural cortical thickness compared with nonisolated control grafts, but this effect was not observed when the periosteum alone was isolated. Quantitative histology performed 10 weeks after surgery indicated that graft isolation was associated with increased marrow fibrosis and necrosis compared with nonisolated controls; it also demonstrated evidence of increased activity in bone remodeling (osteoblast and osteocyte count, new trabecular bone, and surface resorption). Triple fluorochrome staining suggested increased bone turnover in the nonisolated grafts compared with isolated grafts at 1 and 5 weeks postoperatively. This study demonstrates that isolating a rabbit calvarial inlay autogenous bone graft from the dura and/or periosteum results in significantly (p < 0.05) decreased revascularization, interface healing, and cross-sectional areas of amount of mature bone compared with nonisolated control grafts 10 weeks after surgery. At this time point, histologic examination demonstrates a paradoxical increase in bone remodeling in isolated bone grafts compared with controls. It is possible that the inhibition of revascularization results in a delayed onset of the remodeling phase of graft incorporation. However, in the model studied, it is not known whether the quantitative histologic and morphometric parameters measured in these isolated grafts exhibit a "catch-up" phenomenon at time points beyond 10 weeks after surgery. The results of this study emphasize the importance of a healthy recipient site in the healing and incorporation of calvarial bone grafts but stress the need for further investigation at later time points.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

The Effect of Exercise on the Early Stages of Mesenchymal Stromal Cell-Induced Cartilage Repair in a Rat Osteochondral Defect Model

The repair of articular cartilage is challenging owing to the restriction in the ability of articular cartilage to repair itself. Therefore, cell supplementation therapy is possible cartilage repair method. However, few studies have verified the efficacy and safety of cell supplementation therapy. The current study assessed the effect of exercise on early the phase of cartilage repair following cell supplementation utilizing mesenchymal stromal cell (MSC) intra-articular injection. An osteochondral defect was created on the femoral grooves bilaterally of Wistar rats. Mesenchymal stromal cells that were obtained from male Wistar rats were cultured in monolayer. After 4 weeks, MSCs were injected into the right knee joint and the rats were randomized into an exercise or no-exercise intervention group. The femurs were divided as follows: C group (no exercise without MSC injection); E group (exercise without MSC injection); M group (no exercise with MSC injection); and ME group (exercise with MSC injection). At 2, 4, and 8 weeks after the injection, the femurs were sectioned and histologically graded using the Wakitani cartilage repair scoring system. At 2 weeks after the injection, the total histological scores of the M and ME groups improved significantly compared with those of the C group. Four weeks after the injection, the scores of both the M and ME groups improved significantly. Additionally, the scores in the ME group showed a significant improvement compared to those in the M group. The improvement in the scores of the E, M, and ME groups at 8 weeks were not significantly different. The findings indicate that exercise may enhance cartilage repair after an MSC intra-articular injection. This study highlights the importance of exercise following cell transplantation therapy.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.