Purpose: Damaged cartilage has very limited potential for self-repair. Tissue bioengineering offers an interesting alternative for repair of cartilage injury caused by joint trauma or osteochondritis dessicans. The purpose of this work was to use primary chondrocytes cultivated in vitro on collagen gel to produce a neocartilage which can be reimplanted. Material and methods: Chondrocytes were extracted by enzymatic digestion from calf feet harvested from animals aged less than six months. Two million cells were seeded on collagen gels in multiple-well plates and covered with culture medium (1 ml). Type I collagen was acquired from ground calf skin used at a concentration of 1.25 mg/ml. The culture medium was a v/v mixture of RPMI 1640 and NCTC 109. This mixture was supplemented with 10% foetal calf serum, 100 U/ml penicillin, and 250 ng/ml amphotericin B. Cell proliferation was assess fluorometrically and synthesis of glycosaminoglycans (sGAG) by colorimetric assay. Histological study (safranine O) and immunohistochemistry tests (type I and II collagen) were performed to monitor synthesis of matrix components. Expression of genes coding for certain matrix proteins (collagen Ia 2 and 1, II, X, agrecan and MMP13) was studied using RT-PCR. Results: The chondrocyte phenotype was preserved. Type II collagen as well as agrecan was expressed and expression of type I collagen did not increase during the culture. Progressive synthesis of sGAG was observed as was moderate cell proliferation. Cell distribution within the gel was apparently homogeneous. The chondrocytes retained their round shape throughout the study. Type II collagen deposits were visible on day 9 in peripheral cells in areas of high-cell density, then progressed with time. Discussion: Our in vitro results show that three-dimensional cultures of chondrocytes using a collagen gel can produce construction of an extracellular matrix with preservation of chondrocyte phenotype during the culture period. Conclusion: The collagen matrix offers an environment favouring the formation of a functional artificial cartilage by chondrocytes and opens promising perspectives for repairing damaged cartilage

Objectives

We sought to determine if a durable bilayer implant composed of trabecular metal with autologous periosteum on top would be suitable to reconstitute large osteochondral defects. This design would allow for secure implant fixation, subsequent integration and remodeling.

Introduction and Objective. Current cartilage repair strategies lack adequate tissue integration capacity and often present mechanical failure at the graft-to-host tissue junction. The design of multilayered osteochondral tissue engineering (TE) constructs is an attractive approach to overcome these problems. However, calcium ion-release from resorbable bone-replacement materials was suggested to compromise chondrogenic differentiation of adjacent cartilage tissue and it is unclear whether articular chondrocytes (AC) or mesenchymal stroma cells (MSC) are more sensitive to such conditions. Aim of the study was to compare how elevated calcium levels affect cartilage matrix production during re-differentiation of AC versus chondrogenic differentiation of MSC. The results of this study will help to identify the ideal cell source for growth of neocartilage adjacent to a calcified bone replacement material for design of multilayered osteochondral TE approaches. Materials and Methods. Expanded human AC and MSC (6–12 donors per group) were seeded in collagen type I/III scaffolds and cultured under standard chondrogenic conditions at control (1.8mM) or elevated (8.0mM) CaCl2 for 35 days. Proteoglycan and collagen production were assessed via radiolabel-incorporation, ELISA, qPCR and Western blotting. Differences between groups or cell types were calculated using the non-parametric Wilcoxon or Mann-Whitney U test, respectively, with p < 0.05 considered significant. Results. Elevated calcium significantly reduced GAG synthesis (63% of control, p=0.04) and chondrogenic marker expression of AC, lowering the GAG/DNA content (47% of control, p=0.004) and collagen type II deposition (24% of control, p=0.05) of neocartilage compared to control conditions. Opposite, at elevated calcium levels MSC-derived chondrocytes significantly increased GAG synthesis (130% of control, p=0.02) and collagen type II content (160% of control, p=0.03) of cartilage compared to control tissue. Chondrogenic and hypertrophic marker expression was insensitive to calcium levels in MSC-derived chondrocytes. As a result, maturation under elevated calcium allowed for a significantly higher GAG/DNA content in MSC-derived samples compared to AC constructs, although under control conditions both groups developed similarly. Conclusions. AC and MSC showed an opposite reaction to elevation of calcium levels regarding cartilage matrix production and we propose MSC as a preferred cell source to grow chondrocytes in vicinity to calcified bone replacement materials. Since MSC remained prone to hypertrophy under elevated calcium, trizonal cartilage TE constructs, where an AC-layer is separated from the bone replacement phase by an intermediate layer of MSC appear as an ideal design for multilayered osteochondral TE with respect to calcium sensitivity of cells and protection of the upper cartilage layer from hypertrophy

Lesions in the joint surface are commonly treated with osteoarticular autograft transfer system (OATS), autologous cell implantation (ACI/MACI), or microfracture. Tissue formed buy the latter commonly results in mechanically inferior fibrocartilage that fails to integrate with the surrounding native cartilage, rather than durable hyaline cartilage. Fractional laser treatment to make sub-millimeter (<500 µm) channels has been employed for tissue regeneration in the skin to facilitate rejuvenation without typical scarring. Additionally, we have pioneered a means to generate articular cartilage matrix from chondrocytes—dynamic Self-Regenerating Cartilage (dSRC). Combining these two approaches by performing fractional laser treatment of the joint cartilage and treating with dSRC is a new paradigm for joint surface restoration. This approach was refined in a series of in vitro experiments and tested in swine knee defects during a 6-month study in 12 swine. dSRC are generated by placing 10. 7. swine knee chondrocytes into sealed 15-mL polypropylene tubes and cultured on a rocker at 40 cycles per minute for 14 days at 37°C. The chondrocytes aggregate and generate new extracellular matrix to form a pellet of dSRC. Channels of approximately 300-500 µm diameter were created by infrared laser ablation in swine cartilage (in vitro) and swine knees (in vivo). The diameter and depth of the ablated channel in the cartilage was controlled by the light delivery parameters (power, spot size, pulse duration) from a fractional 2.94 µm Erbium laser. The specimens were evaluated with histology (H&E, safranin O, toluidine blue) and polarized-sensitive optical coherence tomography for collagen orientation. We can consistently create laser-ablated channels in the swine knee and successfully implant new cartilage from dSRC to generate typical hyaline cartilage in terms of morphology and biochemical properties. The neocartilage integrates with host cartilage in vivo. These findings demonstrate our novel combinatorial approach for articular cartilage rejuvenation

We investigated the prognostic indicators for collagen-covered autologous chondrocyte implantation (ACI-C) performed for symptomatic osteochondral defects of the knee. We analysed prospectively 199 patients for up to four years after surgery using the modified Cincinnati score. Arthroscopic assessment and biopsy of the neocartilage was also performed whenever possible. The favourable factors for ACI-C include younger patients with higher pre-operative modified Cincinnati scores, a less than two-year history of symptoms, a single defect, a defect on the trochlea or lateral femoral condyle and patients with fewer than two previous procedures on the index knee. Revision ACI-C in patients with previous ACI and mosaicplasties which had failed produced significantly inferior clinical results. Gender (p = 0.20) and the size of the defect (p = 0.97) did not significantly influence the outcome

The treatment of acute full thickness chondral damage within the knee is a surgical challenge. Frequently used surgical techniques include chondroplasty, micro-fracture and chondrocyte implantation. These procedures give unpredictable functional outcomes and if the formation of neocartilage is achieved it is predominantly composed of type 1 collagen. The TruFit osteochondral plug was designed to provide a scaffold for cell proliferation into full thickness chondral defects. It is a composite polymer composed of polylactide co-glycolide, calcium sulphate and poly-glycolide fibres. It is composed of 2 layers, one with a similar trabecular network to cancellous bone and a superficial layer designed to simulate articular lining. The TruFit bone plug was analysed using micro-computed tomography. Its morphology characteristics, granulometry, mechanical performance and image guided failure were tested as well as numerical modelling to assess the permeability of TruFit. Morphological parameters of the TruFit bone plug compared favourably with those of human tissue. Under load the scaffold exhibited shear bands throughout the composite leading to a failure mechanism similar to cancellous bone. Stress relaxation rates of the scaffolds were greatly decreased under wet conditions, likely due to plasticisation of the scaffold by water. The biomechanical properties of the TruFit bone plugs are a cause for concern. The Scaffolds mechanical performance under load rapidly deteriorates in wet conditions at body temperature (the natural knee environment). This early failure will lead to defects in the articular surface where the plug has been inserted. Clinical data is sparse. This study correlates with work performed by Dockery et al & Spalding et al. These clinical studies have shown that the TruFit implant shows no evidence of bone ingrowth or osteoconductivity. It provides no subchondral support to neocartilage or tissue that was stimulated to form around the defects and surgical sites

Mesenchymal stem cells (MSC) are promising for the treatment of articular cartilage defects; however, common protocols for in vitro chondrogenesis induce typical features of hypertrophic chondrocytes reminiscent of endochondral bone formation. This may implicate a risk for graft stability. We here analysed the early healing response in experimental full-thickness cartilage defects, asking whether and how MSC can differentiate to chondrocytes in an orthotopic environment. Cartilage defects in knees of minipigs were covered with a collagen-type I/III membrane, and half of them received transplantation of expanded autologous MSC. Integration into surrounding cartilage tissue was poor to moderate after 1 and 3 weeks and no sign of cartilaginous matrix production as indicated by negative safranin-O staining was visible for both groups. At 8 weeks regenerative tissue was integrated into the surrounding tissue and a safranin-O positively stained neocartilage was detectable in 4 tissue regenerates out of 6 in the MSC group compared to 2 out of 6 in the MSC-free group. At 1 and 3 weeks after surgery only marginal Col2A1 and no AGC expression were detectable in both groups. At 8 weeks Col2A1 and AGC levels had significantly increased. Hypertrophic maker induction (Col10A1 and MMP13) was similar in both groups 8 weeks after surgery. Immunostaining for collagen type X, however, was restricted to the regenerative tissue close to the subchondral bone in both groups, while collagen type II staining was detected from below the superficial to the deep zone. Our data provide molecular evidence for spontaneous differentiation of MSC in cartilage and the development of a collagen type II positive, collagen type X negative neocartilage. Whether by remodelling of defect filling tissue collagen type X positive areas will further diminish or even disappear from repair cartilage at later stages has to be evaluated in a longer follow-up study

Aim of the study was to evaluate if abrasion-arthroplasty (AAP) and abrasion-chondroplasty (ACP) leads to a release of mesenchymal stem cell (MSC) like cells from the bone marrow to the joint cavity where they probably differentiate into a chondrogenic phenotype. Introduction. Cartilage demage is a sever problem in our aging society. About 5 million people only in Germany are affected. Osteoathritis is a degeneration of cartilage caused by aging or traumata 50 % of the people over 40 have signs of osteoarthritis. But the ability of self-regeneration of cartilage is strongly limited. There are different approaches to therapy osteoathritic lesions. Arthroscopic treatment of OA includes bone marrow stimulation technique such as abrasion arthroplasty (AAP) and microfracturing (MF). Beside the support of chondrocyte progenitor cells the environment is also important for the commitment to chondrocytes. Therefore insulin-like growth factor-1 (IGF-1) and transforming growth factor beta-1 (TGF-β1) are important factors during the regeneration process. In the present study we characterised the heamarthrosis and the released cells after AAP and its ability to differentiate into the chondrocyte lineage. Material and Methods. Postoperative haemarthrosis was taken 5, 22 or 44 hours after surgery. 7.5 mg Dexamethasone (Corticosteroid) was administered into the knee joint to prevent postoperative inflammation. Mononuclear cells were isolated from haemarthrosis from the drainage bottle by ficoll density gradient centrifugation. The isolated cells were characterised using fluorescence-activated cell-sorting (FACS) analysis for characteristic markers of MSC such as CD 44, 73, 90, 105. After expanding cells were cultured in a pellet culture. After 3 weeks, histochemistry and immunohistochemistry against Sox9, collagen II and proteoglycan were performed. The release of IGF1, BMP4 and BMP7 was analysed in haemarthrosis serum by ELISA and Luminex technology. Results. The isolated cells after AAP are positive for the mesenchymal stem cell marker CD105, CD90, CD73, CD 44 and negative for the marker of hematopoetic stem cells CD 34. Isolated cells after ACP couldn't be expanded for further characterizations. The staining of the 3D-culture revealed a positive signal for the chondrogen transcription factor Sox9 and the expression of extracellular markerproteins like collagen type II and proteoglycan. Both surgery techniques, AAP and ACP provides a chondrogenic environment. We were able to detect IGF-1, TGFß, BMP4 and BMP7 in the haemarthrosis. Discussion. The benefit of abrasion arthroplasty surgery and microfracturing is controversial discussed because they do not consistently result in hyaline cartilage. But the opening of the bone marrow allows the release of monocytic cells which have the potential to differentiate into a chondrogenic phenotype. In 3D-culture these cells express Sox9 and a collagen proteoglycan rich matrix. The haemarthrosis provides also a cartilage-stimulating environment. We could detect IGF1, TGFβ, BMP4 and 7 which could enhance the commitment concerning differentiation of MSCs to a chondrogenic lineage concerning the production of cartilage specific extracellular matrix. Taken together our study provides the evidence for a therapeutic benefit of opening bone marrow in order to generate neocartilage after AAP

Aims

The purpose of this study was to explore a simple and effective method of preparing human acellular amniotic membrane (HAAM) scaffolds, and explore the effect of HAAM scaffolds with juvenile cartilage fragments (JCFs) on osteochondral defects.

Aims

Extracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold.

The past ten years have brought plenty of research and technical innovations and also preliminary clinical success in cartilage repair. The common target of all methods utilised is to produce a sufficiently stable quality of cartilage repair or regenerate. However, yet today clinical, radiological and histological results analysing the different techniques are somewhat contradictory. The different lines of clinically applied and basic research have focused on:. 1) Spontaneous natural filling of the defect with fibro-cartilage of variable solidity. - Abrasion chondroplasty, drilling or microfracturing to allow for mobilisation of progenitor cells and mesenchymal stem cells from the cancellous bone into the defect and develop to a hyaline like cartilage. - Stem cell treatment (in vivo or ex vivo theory of potential technique by which stem cells could be brought to a defect to create cartilage; so far no directly linked product available). 2) Transplantation of osteochondral auto grafts (Mosaicplasty, OATS, SDS, patellar graft) or allograft. 3a) Autologous chondrocyte transplantation and periosteal coverage (ACT) to cover bigger surfaces. 3b) Implantation of second and third generation ex vivo products and create less morbidity but without knowing whether the results are as long-lasting as for the originally described technique (chondrocytes cultured on membranes, MACI, in gels, implantation of a stable three-dimensional de novo cartilage disk or even engineered osteochondral grafts, AMIC: autologous membrane induced chondrogenesis). A fair amount of today’s laboratory research is focusing on the culture of the patients own chondrocytes or his own stem cells. Clinically, some methods can be applied in all indications regardless of size, localisation, depth of the lesion up to the age of fifty years and this is valid for lesions in the knee, the shoulder, the talus, the elbow etc. Other methods like AOCT should not be used for lesions over 2cm in diameter because of donor side morbidity. All methods claim to have an 85% outcome success rate. Regarding the histological content of the successful implants or the reformed cartilage, microfracturing produces a cartilage implant containing a fibrocartilage that looks similar to the hyaline like cartilage of ACI at two years. Mosaicplasty plugs provided great care is applied during insertion avoiding damage of the cylinders and cartilage death-a special instrumentation has been developed with ZIMMER, the Soft Delivery System, SDS to avoid force during impaction. They remain hyaline provided they are inserted without being prone or deep sunken and the surface convexity of the femoral condyle is restored and provided they are inserted tightly next to each other. There is agreement that this is more difficult in arthroscopic techniques. One agrees also that results are dependent on the alignment of the limb. If the compartment treated is overloaded, there is less chance for integration. Osteotomy has therefore a solid position in the armamentarium of the cartilage surgeon- up to 50% of our cases get an osteotomy as part of their treatment regardless of which technique is utilised. As complications in autologous osteochondral grafting we may observe destruction of the hyaline cartilage cap, non integration and pseudarthrosis or fractures of the cylinders (of special risk are smokers), especially when grafts are not inserted tightly to each other and there is lack of stability with fluid leakage out of the cartilage caps. Rarely ossification of the cartilage is observed when a thin capped cylinder retrieved in the peripheral zone of the femoral trochlea is implanted in an area of thick cartilage as in the centre of the patella where the cartilage is 5 mm thick. Donor site pathology in mosaicplasty is an issue of concern mainly if more than six plugs are removed from the femoropatellar joint. This alone can create clinical symptoms. Nicotine abuse, probably for all techniques decreases the rate of success of cartilage repair or regeneration and osteotomy healing. Roughly 300 cases have been treated during the last 10 years. The results were reported in 2002. As an alternate single surgery technique to microfracturing and mosaicplasty we adopted the “Autologous membrane induced chondrogenesis” (AMIC) technique proposed by Behrens that we find especially useful in OCD. In this relatively young technique we curette the defect and apply microfractures to the basis of the osseous defect. Then we gain cancellous bone from the tibial plateau and mix it with fibrin glue, of which 50% of the thrombin portion is replaced by the serum of the patient as a source of growth factor. This paste of bone and enriched fibrin glue is filled in the defect which is then covered by the porcine Chondrogide membrane (Geistlich) that is glued on and which we can as well suture to the defect. The AMIC technique in combination with microfractures can be utilised for the coverage of pure cartilage defects alone where the membrane is glued alone or fixed on the defect in combination with 5-0 resorbable sutures. In the first two weeks following surgery, after treatment is very defensive to avoid loss of the membrane. After two months of crutch walking with 15 kg of weight we observe a nice osseous integration of the graft and a covering layer that looks promising. After 4–6 months activity can be increased depending on the size of the defect. This is a young technique that we adopted in mid 2003 with 30 cases treated so far, therefore strict observation is required over the upcoming years regarding clinical results and durability and also the composition of this neocartilage. So far it seems to be an interesting alternative to Mosaicplasty since it combines principles of cell therapy with an artificial and instant biological containment that acts against the loss of cells thus acting as a internal bioreactor with the patients own growth factor support

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation.

Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263–273. DOI: 10.1302/2046-3758.74.BJR-2018-0006.

In this study a combination of autologous chondrocyte implantation (ACI) and the osteochondral autograft transfer system (OATS) was used and evaluated as a treatment option for the repair of large areas of degenerative articular cartilage. We present the results at three years post-operatively. Osteochondral cores were used to restore the contour of articular cartilage in 13 patients with large lesions of the lateral femoral condyle (n = 5), medial femoral condyle (n = 7) and patella (n = 1). Autologous cultured chondrocytes were injected underneath a periosteal patch covering the cores. After one year, the patients had a significant improvement in their symptoms and after three years this level of improvement was maintained in ten of the 13 patients. Arthroscopic examination revealed that the osteochondral cores became well integrated with the surrounding cartilage. We conclude that the hybrid ACI/OATS technique provides a promising surgical approach for the treatment of patients with large degenerative osteochondral defects.