Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded in vitro. Approximately 30 million cells per ml of cultured chondrocytes were incorporated with autologous plasma-derived fibrin to form a three-dimensional construct. Full-thickness punch hole defects were created in the lateral and medial femoral condyles. The defects were implanted with either an autologous ‘chondrocyte-fibrin’ construct (ACFC), autologous chondrocytes (ACI) or fibrin blanks (AF) as controls. Animals were killed after 12 weeks. The gross appearance of the treated defects was inspected and photographed. The repaired tissues were studied histologically and by scanning electron microscopy analysis. All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O’Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage. Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI

Human bone-marrow mesenchymal stem cells have an important role in the repair of musculoskeletal tissues by migrating from the bone marrow into the injured site and undergoing differentiation. We investigated the use of autologous human serum as a substitute for fetal bovine serum in the ex vivo expansion medium to avoid the transmission of dangerous transfectants during clinical reconstruction procedures. Autologous human serum was as effective in stimulating growth of bone-marrow stem cells as fetal bovine serum. Furthermore, medium supplemented with autologous human serum was more effective in promoting motility than medium with fetal bovine serum in all cases. Addition of B-fibroblast growth factor to medium with human serum stimulated growth, but not motility. Our results suggest that autologous human serum may provide sufficient ex vivo expansion of human bone-marrow mesenchymal stem cells possessing multidifferentiation potential and may be better than fetal bovine serum in preserving high motility

Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

Our aim was to examine the potential of autologous perichondral tissue to form a meniscal replacement. In 18 mature sheep we performed a complete medial meniscectomy. The animals were then divided into two groups: 12 had a meniscal replacement using strips of autologous perichondral tissue explanted from the lower rib (group G) and six (group C) served as a control group without a meniscal replacement. In all animals restriction from weight-bearing was achieved by means of transection and partial resection of tendo Achillis. Six animals (four from group G and two from group C) were each killed at 3, 6 and 12 months. The grafts and the underlying articular cartilage were removed and studied by gross macroscopic examination, light microscopy, SEM, polarised light examination, and by biomechanical tests. In all the transplanted animals a new perichondral meniscus developed. After three months the transplants resembled normal menisci in size and thickness, while in the control animals only small rims of spontaneously grown tissue were seen. Microscopically, the perichondral menisci showed a normal orientation of collagen fibres and normal cellular characteristics, but in the central region, areas of calcification disturbed the regular tissue differentiation. Healing tissue in control animals lacked the normal fibre orientation and cellularity. SEM of perichondral menisci showed surface characteristics similar to those of normal sheep menisci without fissures and lacerations; the control specimens had these defects. The femoral and tibial cartilage in contact with the new menisci had normal surface characteristics apart from one animal with slight surface irregularities. Control animals showed superficial lesions after three months which increased at six to 12 months postoperatively. Microangiography of the newly grown tissue demonstrated a less intense vascularisation after three months when compared with normal menisci. The failure stress and tensile modulus of perichondral menisci were significantly lower than those of normal contralateral menisci, and spontaneously regenerated tissue in meniscectomised animals had even lower values. There were no significant differences in values between newly grown perichondral menisci and spontaneously grown tissue

Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage. Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage

We studied the effects of irradiation on the reintegration of autologous osteoarticular grafts over a period of 24 weeks in a canine model. In 16 foxhounds the medial femoral condyle was resected, irradiated and immediately replanted. In the control group resection and replantation were performed without irradiation. Reintegration was assessed by macroscopic analysis, histology, radiography and gait analysis. Reintegration was equal at 12 weeks, but significantly inferior in the irradiated group after 24 weeks with delayed bone remodelling. The articular cartilage showed modest degeneration. Conventional radiography and histology showed corresponding changes. Limb function was adequate but the gait was inferior in the treated group

The properties of impacted morsellised bone graft (MBG) in revision total knee arthroplasty (TKA) were studied in 12 horses. The left hind metatarsophalangeal joint was replaced by a human TKA. The horses were then randomly divided into graft and control groups. In the graft group, a unicondylar, lateral uncontained defect was created in the third metatarsal bone and reconstructed using autologous MBG before cementing the TKA. In the control group, a cemented TKA was implanted without the bone resection and grafting procedure. After four to eight months, the animals were killed and a biomechanical loading test was performed with a cyclic load equivalent to the horse’s body-weight to study mechanical stability. After removal of the prosthesis, the distal third metatarsal bone was studied radiologically, histologically and by quantitative and micro CT. Biomechanical testing showed that the differences in deformation between the graft and the control condyles were not significant for either elastic or time-dependent deformations. The differences in bone mineral density (BMD) between the graft and the control condyles were not significant. The BMD of the MBG was significantly lower than that in the other regions in the same limb. Micro CT showed a significant difference in the degree of anisotropy between the graft and host bone, even although the structure of the area of the MBG had trabecular orientation in the direction of the axial load. Histological analysis revealed that all the grafts were revascularised and completely incorporated into a new trabecular structure with few or no remnants of graft. Our study provides a basis for the clinical application of this technique with MBG in revision TKA

Platelet-rich plasma is a new inductive therapy which is being increasingly used for the treatment of the complications of bone healing, such as infection and nonunion. The activator for platelet-rich plasma is a mixture of thrombin and calcium chloride which produces a platelet-rich gel.

We analysed the antibacterial effect of platelet-rich gel in vitro by using the platelet-rich plasma samples of 20 volunteers. In vitro laboratory susceptibility to platelet-rich gel was determined by the Kirby-Bauer disc-diffusion method. Baseline antimicrobial activity was assessed by measuring the zones of inhibition on agar plates coated with selected bacterial strains.

Zones of inhibition produced by platelet-rich gel ranged between 6 mm and 24 mm (mean 9.83 mm) in diameter. Platelet-rich gel inhibited the growth of Staphylococcus aureus and was also active against Escherichia coli. There was no activity against Klebsiella pneumoniae, Enterococcus faecalis, and Pseudomonas aeruginosa. Moreover, platelet-rich gel seemed to induce the in vitro growth of Ps. aeruginosa, suggesting that it may cause an exacerbation of infections with this organism. We believe that a combination of the inductive and antimicrobial properties of platelet-rich gel can improve the treatment of infected delayed healing and nonunion.

The aim of this study was to evaluate the cultivation potential of cartilage taken from the debrided edge of a chronic lesion of the articular surface. A total of 14 patients underwent arthroscopy of the knee for a chronic lesion on the femoral condyles or trochlea. In addition to the routine cartilage biopsy, a second biopsy of cartilage was taken from the edge of the lesion. The cells isolated from both sources underwent parallel cultivation as monolayer and three-dimensional (3D) alginate culture. The cell yield, viability, capacity for proliferation, morphology and the expressions of typical cartilage genes (collagen I, COL1; collagen II, COL2; aggrecan, AGR; and versican, VER) were assessed. The cartilage differentiation indices (COL2/COL1, AGR/VER) were calculated. The control biopsies revealed a higher mean cell yield (1346 cells/mg vs 341 cells/mg), but similar cell proliferation, viability and morphology compared with the cells from the edge of the lesion. The cartilage differentiation indices were superior in control cells: COL2/COL1 (threefold in biopsies (non-significant)); sixfold in monolayer cultures (p = 0.012), and 7.5-fold in hydrogels (non-significant), AGR/VER (sevenfold in biopsies (p = 0.04), threefold (p = 0.003) in primary cultures and 3.5-fold in hydrogels (non-significant)).

Our results suggest that the cultivation of chondrocytes solely from the edges of the lesion cannot be recommended for use in autologous chondrocyte implantation.

Objectives. To explore the therapeutic potential of combining bone marrow-derived mesenchymal stem cells (BM-MSCs) and hydroxyapatite (HA) granules to treat nonunion of the long bone. Methods. Ten patients with an atrophic nonunion of a long bone fracture were selectively divided into two groups. Five subjects in the treatment group were treated with the combination of 15 million autologous BM-MSCs, 5g/cm. 3. (HA) granules and internal fixation. Control subjects were treated with iliac crest autograft, 5g/cm. 3. HA granules and internal fixation. The outcomes measured were post-operative pain (visual analogue scale), level of functionality (LEFS and DASH), and radiograph assessment. Results. Post-operative pain evaluation showed no significant differences between the two groups. The treatment group demonstrated faster initial radiographic and functional improvements. Statistically significant differences in functional scores were present during the first (p = 0.002), second (p = 0.005) and third (p = 0.01) month. Both groups achieved similar outcomes by the end of one-year follow-up. No immunologic or neoplastic side effects were reported. Conclusions. All cases of nonunion of a long bone presented in this study were successfully treated using autologous BM-MSCs. The combination of autologous BM-MSCs and HA granules is a safe method for treating nonunion. Patients treated with BM-MSCs had faster initial radiographic and functional improvements. By the end of 12 months, both groups had similar outcomes. Cite this article: H.D. Ismail, P. Phedy, E. Kholinne, Y. P. Djaja, Y. Kusnadi, M. Merlina, N. D. Yulisa. Mesenchymal stem cell implantation in atrophic nonunion of the long bones: A translational study. Bone Joint Res 2016;5:287–293. DOI: 10.1302/2046-3758.57.2000587

Implantation of autologous chondrocytes and matrix autologous chondrocytes are techniques of cartilage repair used in the young adult knee which require harvesting of healthy cartilage and which may cause iatrogenic damage to the joint. This study explores alternative sources of autologous cells. Chondrocytes obtained from autologous bone-marrow-derived cells and those from the damaged cartilage within the lesion itself are shown to be viable alternatives to harvest-derived cells. A sufficient number and quality of cells were obtained by the new techniques and may be suitable for autologous chondrocyte and matrix autologous chondrocyte implantation

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. Materials and Methods. Adult sheep were randomly assigned to one of three groups (n = 8/group): 1. trabecular metal/periosteal graft (TMPG), 2. trabecular metal (TM), 3. empty defect (ED). Cartilage and bone healing were assessed macroscopically, biochemically (type II collagen, sulfated glycosaminoglycan (sGAG) and double-stranded DNA (dsDNA) content) and histologically. Results. At 16 weeks post-operatively, histological scores amongst treatment groups were not statistically different (TMPG: overall 12.7, cartilage 8.6, bone 4.1; TM: overall 14.2, cartilage 9.5, bone 4.9; ED: overall 13.6, cartilage 9.1, bone 4.5). Metal scaffolds were incorporated into the surrounding bone, both in TM and TMPG. The sGAG yield was lower in the neo-cartilage regions compared with the articular cartilage (AC) controls (TMPG 20.8/AC 39.5, TM 25.6/AC 33.3, ED 32.2/AC 40.2 µg sGAG/1 mg respectively), with statistical significance being achieved for the TMPG group (p < 0.05). Hypercellularity of the neo-cartilage was found in TM and ED, as the dsDNA content was significantly higher (p < 0.05) compared with contralateral AC controls (TM 126.7/AC 71.1, ED 99.3/AC 62.8 ng dsDNA/1 mg). The highest type II collagen content was found in neo-cartilage after TM compared with TMPG and ED (TM 60%/TMPG 40%/ED 39%). Inter-treatment differences were not significant. Conclusions. TM is a highly suitable material for the reconstitution of osseous defects. TM enables excellent bony ingrowth and fast integration. However, combined with autologous periosteum, such a biocomposite failed to promote satisfactory neo-cartilage formation. Cite this article: E. H. Mrosek, H-W. Chung, J. S. Fitzsimmons, S. W. O’Driscoll, G. G. Reinholz, J. C. Schagemann. Porous tantalum biocomposites for osteochondral defect repair: A follow-up study in a sheep model. Bone Joint J 2016;5:403–411. DOI: 10.1302/2046-3758.59.BJR-2016-0070.R1

Objectives. Mesenchymal stem cells have the ability to differentiate into various cell types, and thus have emerged as promising alternatives to chondrocytes in cell-based cartilage repair methods. The aim of this experimental study was to investigate the effect of bone marrow derived mesenchymal stem cells combined with platelet rich fibrin on osteochondral defect repair and articular cartilage regeneration in a canine model. Methods. Osteochondral defects were created on the medial femoral condyles of 12 adult male mixed breed dogs. They were either treated with stem cells seeded on platelet rich fibrin or left empty. Macroscopic and histological evaluation of the repair tissue was conducted after four, 16 and 24 weeks using the International Cartilage Repair Society macroscopic and the O’Driscoll histological grading systems. Results were reported as mean and standard deviation (. sd. ) and compared at different time points between the two groups using the Mann-Whitney U test, with a value < 0.05 considered statistically significant. Results. Higher cumulative macroscopic and histological scores were observed in stem cell treated defects throughout the study period with significant differences noted at four and 24 weeks (9.25, . sd. 0.5 vs 7.25, . sd. 0.95, and 10, . sd. 0.81 vs 7.5, . sd. 0.57; p < 0.05) and 16 weeks (16.5, . sd. 4.04 vs 11, . sd. 1.15; p < 0.05), respectively. Superior gross and histological characteristics were also observed in stem cell treated defects. Conclusion. The use of autologous culture expanded bone marrow derived mesenchymal stem cells on platelet rich fibrin is a novel method for articular cartilage regeneration. It is postulated that platelet rich fibrin creates a suitable environment for proliferation and differentiation of stem cells by releasing endogenous growth factors resulting in creation of a hyaline-like reparative tissue. Cite this article: D. Kazemi, K. Shams Asenjan, N. Dehdilani, H. Parsa. Canine articular cartilage regeneration using mesenchymal stem cells seeded on platelet rich fibrin: Macroscopic and histological assessments. Bone Joint Res 2017;6:98–107. DOI: 10.1302/2046-3758.62.BJR-2016-0188.R1

Objectives. To compare the therapeutic potential of tissue-engineered constructs (TECs) combining mesenchymal stem cells (MSCs) and coral granules from either Acropora or Porites to repair large bone defects. Materials and Methods. Bone marrow-derived, autologous MSCs were seeded on Acropora or Porites coral granules in a perfusion bioreactor. Acropora-TECs (n = 7), Porites-TECs (n = 6) and bone autografts (n = 2) were then implanted into 25 mm long metatarsal diaphyseal defects in sheep. Bimonthly radiographic follow-up was completed until killing four months post-operatively. Explants were subsequently processed for microCT and histology to assess bone formation and coral bioresorption. Statistical analyses comprised Mann-Whitney, t-test and Kruskal–Wallis tests. Data were expressed as mean and standard deviation. Results. A two-fold increaseof newly formed bone volume was observed for Acropora-TECs when compared with Porites-TECs (14 . sd. 1089 mm. 3. versus 782 . sd. 507 mm. 3. ; p = 0.09). Bone union was consistent with autograft (1960 . sd. 518 mm. 3. ). The kinetics of bioresorption and bioresorption rates at four months were different for Acropora-TECs and Porites-TECs (81% . sd. 5% versus 94% . sd. 6%; p = 0.04). In comparing the defects that healed with those that did not, we observed that, when major bioresorption of coral at two months occurs and a scaffold material bioresorption rate superior to 90% at four months is achieved, bone nonunion consistently occurred using coral-based TECs. Discussion. Bone regeneration in critical-size defects could be obtained with full bioresorption of the scaffold using coral-based TECs in a large animal model. The superior performance of Acropora-TECs brings us closer to a clinical application, probably because of more suitable bioresorption kinetics. However, nonunion still occurred in nearly half of the bone defects. Cite this article: A. Decambron, M. Manassero, M. Bensidhoum, B. Lecuelle, D. Logeart-Avramoglou, H. Petite, V. Viateau. A comparative study of tissue-engineered constructs from Acropora and Porites coral in a large animal bone defect model. Bone Joint Res 2017;6:208–215. DOI: 10.1302/2046-3758.64.BJR-2016-0236.R1

The gelatin-based haemostyptic compound Spongostan was tested as a three-dimensional (3D) chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. In a control experiment of monolayer cultures, the proliferation or de-differentiation of bovine chondrocytes was either not or only marginally influenced by the presence of Spongostan (0.3 mg/ml). In monolayers and 3-D Minusheet culture chambers, the cartilage-specific differentiation markers aggrecan and type-II collagen were ubiquitously present in a cell-associated fashion and in the pericellular matrix. The Minusheet cultures usually showed a markedly higher mRNA expression than monolayer cultures irrespective of whether Spongostan had been present or not during culture. Although the de-differentiation marker type-I collagen was also present, the ratio of type-I to type-II collagen or aggrecan to type-I collagen remained higher in Minusheet 3-D cultures than in monolayer cultures irrespective of whether Spongostan had been included in or excluded from the monolayer cultures. The concentration of GAG in Minusheet cultures reached its maximum after 14 days with a mean of 0.83 ± 0.8 μg/10. 6. cells; mean ±, . sem. , but remained considerably lower than in monolayer cultures with/without Spongostan. Our results suggest that Spongostan is in principle suitable as a 3-D chondrocyte matrix, as demonstrated in Minusheet chambers, in particular for a culture period of 14 days. Clinically, differentiating effects on chondrocytes, simple handling and optimal formability may render Spongostan an attractive 3-D scaffold for autologous chondrocyte transplantation

The feasibility of bone transport with bone substitute and the factors which are essential for a successful bone transport are unknown. We studied six groups of 12 Japanese white rabbits. Groups A to D received cylindrical autologous bone segments and groups E and F hydroxyapatite prostheses. The periosteum was preserved in group A so that its segments had a blood supply, cells, proteins and scaffold. Group B had no blood supply. Group C had proteins and scaffold and group D had only scaffold. Group E received hydroxyapatite loaded with recombinant human bone morphogenetic protein-2 and group F had hydroxyapatite alone. Distraction osteogenesis occurred in groups A to C and E which had osteo-conductive transport segments loaded with osteo-inductive proteins. We conclude that scaffold and proteins are essential for successful bone transport, and that bone substitute can be used to regenerate bone

We evaluated the histological changes before and after fixation in ten knees of ten patients with osteochondritis dissecans who had undergone fixation of the unstable lesions. There were seven males and three females with a mean age of 15 years (11 to 22). The procedure was performed either using bio-absorbable pins only or in combination with an autologous osteochondral plug. A needle biopsy was done at the time of fixation and at the time of a second-look arthroscopy at a mean of 7.8 months (6 to 9) after surgery. The biopsy specimens at the second-look arthroscopy showed significant improvement in the histological grading score compared with the pre-fixation scores (p < 0.01). In the specimens at the second-look arthroscopy, the extracellular matrix was stained more densely than at the time of fixation, especially in the middle to deep layers of the articular cartilage. Our findings show that articular cartilage regenerates after fixation of an unstable lesion in osteochondritis dissecans

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

We produced large full-thickness articular cartilage defects in 33 rabbits in order to evaluate the effect of joint distraction and autologous culture-expanded bone-marrow-derived mesenchymal cell transplantation (ACBMT) at 12 weeks. After fixing the knee on a hinged external fixator, we resected the entire surface of the tibial plateau. We studied three groups: 1) with and without joint distraction; 2) with joint distraction and collagen gel, and 3) with joint distraction and ACBMT and collagen gel. The histological scores were significantly higher in the groups with ACBMT collagen gel (p < 0.05). The area of regenerated soft tissue was smaller in the group allowed to bear weight (p < 0.05). These findings suggest that the repair of large defects of cartilage can be enhanced by joint distraction, collagen gel and ACBMT