Cartilage tissue engineering can provide substantial relief to people suffering from degenerative cartilage disease, such as osteoarthritis. The autologous platelet-rich plasma (PRP) application appears to improve cartilage healing due to its ability to positively influence cellular mechanisms, mainly in cells from synovium and cartilage. Primary cultures of human synovial fluid stem cells (synoviocytes, SCs) and chondrocytes (CCs) were exposed to various concentrations of non-activated PRP and plateletpoor plasma (PPP) prepared by apheresis. Cell proliferation and migration were evaluated in real-time with the non-invasive xCELLigence System. It was found that PRP had a similar effect on the growth of cells as fetal bovine serum (FBS). Surprisingly, our proliferation assay results indicated that 50% PPP had the largest effect on both cell types, with a statistically significant increase in cell number (P<0.001) compared to the (0% FBS) in vitro control. The migratory ability of SCs was significantly enhanced with 10% PRP and 0.8% hyaluronic acid (HA). HA also augmented migration of CCs. In summary, these results demonstrate that directed cell proliferation and migration are inducible in human articular CCs and SCs, and that both platelet-derived fractions may exert a positive effect and modulate several cell responses that are potentially involved in tissue integration during cartilage repair.

Objective This study aimed to compare microfracture and application of adipose-derived stem cells (ADSCs) by local adherent technique enhanced by platelet-rich plasma (PRP) to provide a new approach for the repair of cartilage defect. Design Full-thickness cylindrical defects were created in the medial femoral condyle in 9 New Zealand White rabbits (5 months old, 4.65 ± 0.20 kg). Two groups of rabbits ( n = 3) were either treated with ADSCs (Group 1) or the microfracture technique (Group 2) following intraarticular injection of PRP 3 times in weekly intervals. Rabbits in control group ( n = 3) remained untreated. The outcome was assessed macroscopically, histologically, and immunohistochemically. Results At the end of week 12, Group 1 showed better defect filling compared with Group 2. Specimens treated with the combination of ADSCs and PRP exhibited significant differences from the other groups in all criteria of International Cartilage Repair Society macroscopic scoring system. Conclusions Intraarticular injection of autologous PRP in combination with transplantation of autologous ADSCs by local adherent technique enhances the quality of cartilage defect repair with better results in comparison with microfracture surgery in a rabbit model.

The topic of the study was to verify in vivo survival of in vitro cultured autologous and allogenous chondrocytes suspended in a fibrin glue Beriplast® or seeded on Collagen type I-Hyaluronan (Col type I-HYA) scaffolds for the regeneration of articular cartilage defects in rabbits. The study was carried out on 15 domestic rabbits randomly assigned to five groups (n = 3 in each) with different treatments of artificially created chondral defects (ChD´s). These defects were made in a non-load-bearing area of medial condyle of the distal femur, and were treated as follows: 1st and 3rd group: the ChD´s were filled with autologous or allogenous chondrocytes seeded on Col type I-HYA scaffolds, respectively. The scaffolds were fixed to the ChD´s by fibrin glue Beriplast®; 2nd and 4th group: the ChD´s were filled with a suspension of autologous or allogenous chondrocytes in fibrin glue Beriplast®, respectively, and they were immediately covered by unseeded Col type I-HYA scaffolds; Control group: the ChD´s were left to heal spontaneously without any treatment. Macroscopical, histological and immunohistochemical analyses of the ChD´s were performed 12 months after the treatment. In all treated groups, the chondrocytes were capable to proliferate and produce the cartilage extracellular matrix, including proteoglycans and type II collagen, as compared to the control “untreated” group. On the other hand, the production of hyaline-like cartilage tissue confirmed that both therapeutic methods using autologous chondrocytes can be applied successfully for the treatment of chondral defects in rabbits.

Avian osteoblasts have been isolated particularly from chicken embryo, but data about other functional tissue sources of adult avian osteoblast precursors are missing. The method of preparation of pigeon osteoblasts is described in this study. We demonstrate that pigeon cancellous bone derived osteoblasts have particular proliferative capacity in vitro in comparison to mammalian species and developed endogenous ALP. Calcium deposits formation in vitro was confirmed by alizarin red staining. Only a few studies have attempted to investigate bone grafting and treatment of bone loss in birds. Lack of autologous bone grafts in birds has prompted investigation into the use of avian xenografts for bone augmentation. Here we present a method of xenografting of ostrich demineralised cancellous bone scaffold seeded with allogeneic adult pigeon osteoblasts. Ostrich demineralised cancellous bone scaffold supported proliferation of pigeon osteoblasts during two weeks of co - cultivation in vitro. Scanning electron microscopy demonstrated homogeneous adult pigeon osteoblasts attachment and distribution on the surface of xenogeneic ostrich demineralised cancellous bone. Our preliminary in vitro results indicate that demineralised cancellous bone from ostrich tibia could provide an effective biological support for growth and proliferation of allogeneic osteoblasts derived from cancellous bone of pigeons.

Basics of isolation and cultivation of chondrocytes according to good laboratory practice ResumenObjetivos: El objetivo del presente estudio era determinar si los condrocitos aislados de cinco pacientes ancianos (edad media 63 años) con artrosis (grado 3) mantienen su proliferación y potencial condrogénico. El aislamiento y cultivo de condrocitos fueron llevados a cabo de acuerdo a los estándares de buenas prácticas de laboratorio. Métodos: Los condrocitos fueron aislados de una biopsia de cartílago mediante digestión enzimática. El cultivo fue llevado a cabo en un ambiente controlado (sala blanca). La caracterización del fenotipo de los condrocitos se logró mediante análisis de citometría de flujo. Resultados: Tras tres semanas de cultivo se podían observar estructuras poligonales propias de los condrocitos, pero también se observaba morfología de tipo fibroblasto en el cultivo. El análisis de la citometría de flujo reveló que el fenotipo de los condrocitos cultivados tras el primer pasaje era positivo para CD44 (98,92%), CD90 (97,11%) y negativo para el marcador hematopoyético CD45 (0,10%). Conclusiones: Los condrocitos articulares humanos obtenidos de cinco pacientes ancianos con artrosis mantenían un fenotipo condrocitario y podrían ser potencialmente utilizados para la implantación autóloga. Las condiciones para el cultivo fueron establecidas de acuerdo a los estándares de buenas prácticas de laboratorio para así minimizar el riesgo de contaminación celular in vitro. AbstractObjectives: The objective of the present study was to determine if chondrocytes isolated from human cartilage of five elderly patients (middle age 63) with osteoarthritis (stage 3) maintain their proliferation and chondrogenic potential. Isolation and cultivation of chondrocytes was performed according to good laboratory practice (GLP) standards. Methods: Chondrocytes were isolated from cartilage biopsy by enzymatic digestion. Cultivation of cells was performed in a controlled environment (cleanroom). Phenotype characterization of chondrocytes was achieved by flow cytometry analysis. Results: Three weeks after cultivation polygonal structures typical for chondrocytes were observed, but spindle/fibroblast like morphology was also detected in culture. Flow cytometric analysis showed that chondrocytes were positive for CD44 (98,35% ± 0,50), CD90 (97,15% ± 0,13) after first passage (P1) and the cells were negative for hematopoietic marker CD45 (0,21% ± 0,11). Conclusions: Human articular chondrocytes obtained from five elderly patients with osteoarthritis maintained a chondrocyte phenotype and could be potentially used for autologous implantation. We have standardized the conditions for cultivation according to GLP standards to minimize the risk of in vitro cell contamination.