Regeneration Of Cartilage Defects Research Articles

An in situ photocrosslinkable platelet rich plasma - Complexed hydrogel glue with growth factor controlled release ability to promote cartilage defect repair.

In this study, we have developed a photocrosslinkable platelet rich plasma (PRP) - complexed hydrogel glue (HNPRP) for cartilage regeneration. The in situ formed HNPRP hydrogel glue showed not only the controlled release ability of growth factors, but also strong tissue adhesiveness, which could resolve the current problems in clinical application of PRP. Furthermore, HNPRP hydrogel glue could promote integrative hyaline cartilage regeneration, and its reparative efficacy for cartilage defect was better than thrombin activated PRP gel. This study provided not only an effective repair material for cartilage regeneration, but also developed an advanced method for PRP application.

Regeneration Mechanism of Full Thickness Cartilage Defect Using Combination of Freeze Dried Bovine Cartilage Scaffold - Allogenic Bone Marrow Mesenchymal Stem Cells - Platelet Rich Plasma Composite (SMPC) Implantation

Cartilage defect has become serious problem for orthopaedic surgeon and patients because of its difficult healing that might occur when articular cartilage damage never reach subchondral layer. In this study, we used combination of freeze dried bovine cartilage (FDBC) scaffold, bone marrow mesenchymal stem cells (BM-MSCs), and platelet rich plasma (PRP) composite (SMPC) implanted in full thickness cartilage defect. This study is to explain its regeneration mechanism. This is true experimental research with post-test only control group design using New Zealand White Rabbit. 50 rabbits is divided into three groups of SMPC, BM-MSCs and FDBC. 37 rabbits evaluated after twelve weeks. Histopathologic examination showed the number of chondrocytes, collagen thickness and cartilage width are highest on SMPC group. Immunohistochemical examination showed SMPC group has the highest number of chondroprogenitor cells express FGF-2R, Sox-9, and MAPK. Brown Forsythe test resulted in significant increase the number of chondrocytes (p=0,010), collagen thickness (p=0,000), and cartilage surface width (p=0,015), and increase FGF-2R (p=0,000), MAPK (p=0,000), and Sox-9 (p=0,000) on SMPC group. Using path analysis, there is strong influence from FGF-2R, MAPK, and Sox-9 to the increase of chondrocytes, collagen thickness, and cartilage surface width. Hence, SMPC implantation mechanism of full thickness cartilage defect regeneration can be explained.

The Basic Science of Bone Marrow Aspirate Concentrate in Chondral Injuries.

There has been great interest in bone marrow aspirate concentrate (BMAC) as a cost effective method in delivering mesenchymal stem cells (MSCs) to aid in the repair and regeneration of cartilage defects. Alongside MSCs, BMAC contains a range of growth factors and cytokines to support cell growth following injury. However, there is paucity of information relating to the basic science underlying BMAC and its exact biological role in supporting the growth and regeneration of chondrocytes. The focus of this review is the basic science underlying BMAC in relation to chondral damage and regeneration.

Self-Assembled Infrapatellar Fat-Pad Progenitor Cells on a Poly-ε-Caprolactone Film For Cartilage Regeneration.

Cartilage defects resulting from osteoarthritis (OA) or physical injury can severely reduce the quality of life for sufferers. Current treatment options are costly and not always effective in producing stable hyaline cartilage. Here we investigated a new treatment option that could potentially repair and regenerate damaged cartilage tissue. This novel approach involves the application of infrapatellar fat-pad derived chondroprogenitor cells onto a mechanically stable biodegradable polymer film that can be easily implanted into a defect site. Poly-ε-caprolactone (PCL) films were fabricated via solvent casting in either acetone or chloroform. The hydrophobicity, mechanical properties, and surface morphology of the films were examined. Progenitor cells from infrapatellar fat-pad were isolated, expanded, and then seeded onto the films. The cells were allowed to self-assemble on films, and these were then cultured in a chemically defined chondrogenic media for 28 days. The self-assembled tissue was characterized via histological staining, gene expression analysis, immunohistochemistry, and biochemical analysis. Chondrogenic differentiation was induced to generate a cartilaginous matrix upon the films. Despite differences between in the appearance, surface morphology, and mechanical properties of the films cast in chloroform or acetone, both methods produced tissues rich in sulfated glycosaminoglycan and collagen, although the extracellular matrix produced on chloroform-cast films appeared to contain more collagen type II and less collagen type I than acetone-cast films. These self-assembled constructs have the potential to be implanted into defect sites as a potential treatment for cartilage defect regeneration.

High Field Sodium MRI Assessment of Stem Cell Chondrogenesis in a Tissue-Engineered Matrix.

The development of non-invasive assessment techniques in vitro and in vivo is essential for monitoring and evaluating the growth of engineered cartilage tissues. Magnetic resonance imaging (MRI) is the leading non-invasive imaging modality used for assessing engineered cartilage. Typical MRI uses water proton relaxation times (T1 and T2) and apparent diffusion coefficient (ADC) to assess tissue growth. These techniques, while excellent in providing the first assurance of tissue growth, are unspecific to monitor the progress of engineered cartilage extracellular matrix components. In the current article, we present high field (11.7 T, (1)H freq. = 500 MHz) sodium MRI assessment of tissue-engineered cartilage at the early stage of tissue growth in vitro. We observed the chondrogenesis of human bone marrow derived stromal cells seeded in a gradient polymer-hydrogel matrix made out of poly(85 lactide-co-15 glycolide)--PuraMatrix™ for 4 weeks. We calculated the sodium concentration in the engineered constructs using a model of sodium MRI voxels that takes into account scaffold volume, cell density and amount of glycosaminoglycan (GAG). The sodium concentration was then converted to the fixed charge density (FCD) and compared with FCD derived from biochemical GAG analysis. Despite the small amount of GAG present in the engineered constructs, the sodium MRI derived FCD is found to be correlated (Pearson correlation coefficient R = 0.79) with the FCD derived from biochemical analysis. We conclude that sodium MRI could prove to be an invaluable tool in assessing engineered cartilage quantitatively during the repair or regeneration of cartilage defects.

Intermittent PTHrP(1-34) exposure augments chondrogenesis and reduces hypertrophy of mesenchymal stromal cells.

Phenotype instability and premature hypertrophy prevent the use of human mesenchymal stromal cells (MSCs) for cartilage regeneration. Aim of this study was to investigate whether intermittent supplementation of parathyroid hormone-related protein (PTHrP), as opposed to constant treatment, can beneficially influence MSC chondrogenesis and to explore molecular mechanisms below catabolic and anabolic responses. Human MSCs subjected to chondrogenic induction in high-density culture received PTHrP(1-34), forskolin, dbcAMP, or PTHrP(7-34) either constantly or via 6-h pulses (three times weekly), before proteoglycan, collagen type II, and X deposition; gene expression; and alkaline phosphatase (ALP) activity were assessed. While constant application of PTHrP(1-34) suppressed chondrogenesis of MSCs, pulsed application significantly increased collagen type 2 (COL2A1) gene expression and the collagen type II, proteoglycan, and DNA content of pellets after 6 weeks. Collagen type 10 (COL10A1) gene expression was little affected but Indian hedgehog (IHH) expression and ALP activity were significantly downregulated by pulsed PTHrP. A faster response to PTHrP exposure was recorded for ALP activity over COL2A1 regulation, suggesting that signal duration is critical for catabolic versus anabolic reactions. Stimulation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling by forskolin reproduced major effects of both treatment modes, whereas application of PTHrP(7-34) capable of protein kinase C (PKC) signaling was ineffective. Pulsed PTHrP exposure of MSCs stimulated chondrogenesis and reduced endochondral differentiation apparently uncoupling chondrogenic matrix deposition from hypertrophic marker expression. cAMP/PKA was the major signaling pathway triggering the opposing effects of both treatment modes. Intermittent application of PTHrP represents an important novel means to improve chondrogenesis of MSCs and may be considered as a supporting clinical-treatment mode for MSC-based cartilage defect regeneration.

Adipose‐derived mesenchymal stem cells for cartilage tissue engineering: State‐of‐The‐Art inin vivostudies

Several therapeutic approaches have been developed to address hyaline cartilage regeneration, but to date, there is no universal procedure to promote the restoration of mechanical and functional properties of native cartilage, which is one of the most important challenges in orthopedic surgery. For cartilage tissue engineering, adult mesenchymal stem cells (MSCs) are considered as an alternative cell source to chondrocytes. Since little is known about adipose-derived mesenchymal stem cell (ADSC) cartilage regeneration potential, the aim of this review was to give an overview of in vivo studies about the chondrogenic potential and regeneration ability of culture-expanded ADSCs when implanted in heterotopic sites or in osteoarthritic and osteochondral defects. The review compares the different studies in terms of number of implanted cells and animals, cell harvesting sites, in vitro expansion and chondrogenic induction conditions, length of experimental time, defect dimensions, used scaffolds and post-explant analyses of the cartilage regeneration. Despite variability of the in vivo protocols, it seems that good cartilage formation and regeneration were obtained with chondrogenically predifferentiated ADSCs (1 × 10(7) cells for heterotopic cartilage formation and 1 × 10(6) cells/scaffold for cartilage defect regeneration) and polymeric scaffolds, even if many other aspects need to be clarified in future studies.

Mesenchymal stem cells for the treatment of cartilage lesions: from preclinical findings to clinical application in orthopaedics

The aim of this systematic review is to examine the available clinical evidence in the literature to support mesenchymal stem cell (MSC) treatment strategies in orthopaedics for cartilage defect regeneration. The research was performed on the PubMed database considering the English literature from 2002 and using the following key words: cartilage, cartilage repair, mesenchymal stem cells, MSCs, bone marrow concentrate (BMC), bone marrow-derived mesenchymal stem cells, bone marrow stromal cells, adipose-derived mesenchymal stem cells, and synovial-derived mesenchymal stem cells. The systematic research showed an increasing number of published studies on this topic over time and identified 72 preclinical papers and 18 clinical trials. Among the 18 clinical trials identified focusing on cartilage regeneration, none were randomized, five were comparative, six were case series, and seven were case reports; two concerned the use of adipose-derived MSCs, five the use of BMC, and 11 the use of bone marrow-derived MSCs, with preliminary interesting findings ranging from focal chondral defects to articular osteoarthritis degeneration. Despite the growing interest in this biological approach for cartilage regeneration, knowledge on this topic is still preliminary, as shown by the prevalence of preclinical studies and the presence of low-quality clinical studies. Many aspects have to be optimized, and randomized controlled trials are needed to support the potential of this biological treatment for cartilage repair and to evaluate advantages and disadvantages with respect to the available treatments. IV.

Repair of an osteochondral defect by sustained delivery of BMP-2 or TGFβ1 from a bilayered alginate-PLGA scaffold

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor-β1 (TGFβ1) and bone morphogenetic protein-2 (BMP-2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone-orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP-2; and (d) microspheres containing 5 µg BMP-2. Release kinetics and tissue distributions were determined using iodinated ((125) I) GFs. Bioactivity of in vitro released BMP-2 and TGFβ1 was confirmed in cell-based assays. In vivo release profiles indicated good GF release control. 20% of BMP-2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF-treated specimens, there being especially relevant short-term outcomes with 50 ng TGFβ1 and 5 µg BMP-2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP-2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen-type II and aggrecan immunoreactivity 2 weeks post-implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks.

Treatment of chondral defects of the knee with one step matrix-assisted technique enhanced by autologous concentrated bone marrow: In vitro characterisation of mesenchymal stem cells from iliac crest and subchondral bone

Cartilage repair is still an unsolved problem. In the last years many cell-based treatments have been proposed, in order to obtain good regeneration of cartilage defects. The Autologous Matrix-Induced Chondrogenesis technique (AMIC ®) combines the micro-fracture procedure with the use of a specific biological membrane. The phenotypic feature of bone marrow cell population, harvested from iliac crest and knee subchondral bone of patients treated with the AMIC ® technique, enhanced by autologous concentrated bone marrow, was analysed to evaluate potential variations of the cell population. Samples of eleven patients, with isolated chondral lesions grade III or IV were treated with the AMIC ® technique, enhanced by the use of autologous concentrated bone marrow. A small fraction of bone marrow samples, both from iliac crest and from the created micro-fractures, was analysed by FACS analysis and then cultured to verify their proliferative and differentiation potential. An average of 0.04% of concentrated bone marrow cells harvested from the iliac crest, presented mesenchymal stem cell phenotype (CD34 −/CD45 low/CD271 high), whereas just 0.02% of these cells were identified from the samples harvested during the creation of micro-fractures at the knee. After two passages in culture, cells expressed a peculiar profile for MSC. Only MSC from bone marrow could be long-term propagated and were able to efficiently differentiate in the cultures. Although the AMIC ® approach has many advantages, the surgical technique in the application of the microfracture technique remains essential and affects the final result.

Perspectives for the tissue engineering of cartilage from a biological and biomaterial point of view.

Tissue engineering has become a fast growing interdisciplinary branch of research at the interface between life and engineering sciences with important clinical end-points. In this context the regeneration of articular cartilage represents an exciting challenge since hyaline cartilage has a limited capacity for self-repair. Today the use of different scaffold materials combined with in vitro expanded chondrocytes and signalling molecules poses great hopes for an optimal treatment of articular cartilage defects. However, until today the optimal construct of scaffolds, cells and signalling molecules has not yet been found. Since repair and regeneration recapitulate in part ontogenetic processes, the present paper summarizes the regulative mechanisms of endochondral ossification in the growth plate of the long bones to identify possible new signalling molecules for the improvement of tissue engineering-based solutions in the treatment of cartilage defects. The growth plate represents a highly organized structure of chondrocytes and extracellular matrix components in distinguishable proliferation and differentiation stages. It is regulated by various paracrine and hormonal factors. In a second part we present actual trends in scaffold design based on synthetic polymers and natural polymers, stressing their potential use in the regeneration of cartilage defects from the point of view of bioactivity and biocompatibility. In conclusion, both new signalling molecules from basic research and innovative scaffold materials with variable physico-chemical properties open up new and interesting perspectives for the research in optimized tissue engineeredbased therapeutic strategies to treat cartilage defects.

P246 In situ Tissue Engineering - cell-free regeneration of cartilage defects

P246 In situ Tissue Engineering - cell-free regeneration of cartilage defects

Gene-Activated Matrices for Cartilage Defect Reparation

A cartilage defect has a very limited ability to repair itself spontaneously due to the shortage of blood. Many attempts have been made to restore the integrity of cartilage in clinical and experimental studies. Recently, tissue engineering has emerged as a new protocol for lost tissue regeneration. Meanwhile, the defect-repairing environment can be improved by gene therapy methods. Gene-activated matrices (GAM) fabricated with biomaterials and plasmids fill the cartilage defects to restore the integrity of joint surface, facilitating repair cell adhesion and proliferation as well as the synthesis of extracelluar matrix. And they also serve as a local gene delivery system, inducing therapeutic agent expression at the repair site. In the present study, we fabricated two- and three-dimensional matrices from chitosan and gelatin, then added a plasmid DNA encoding transforming growth factors-ss1 (TGF-ss1) for cartilage defect regeneration. First, we demonstrated primary chondrocytes could maintain their biological characteristics and secrete therapeutic proteins when they were cultured onto GAM in vitro. Subsequently we inserted three-dimensional GAM into cartilage defects of rabbit knee joints. With the results of the new cartilage tissue formation, we came to the conclusion that GAM was helpful for new tissue production and this therapeutic protocol provided a cheap, simple, and effective method for cartilage defect reparation.

Cartilage defect regeneration by ex-vivo engineered autologous micro-tissue

Cartilage defect regeneration by ex-vivo engineered autologous micro-tissue

Implantation de chondrocytes autologues pour lésions cartilagineuses du sujet jeune. Étude de 24 cas*

Study aim The aim of this study was to describe the treatment of symptomatic knee cartilage defects on young active patients by autologous chondrocyte implantation and to report preliminary results in 24 patients. Patients and methods Since April 1996, 24 selected patients underwent 25 implantations in five military hospitals. There were 19 men and five women (all of them practicing sports); mean age was 27. Lesions were localized on left ( n = 13) and right ( n = 12) aligned and stabilized knees. There were 12 isolated cartilage defects (eight OCD and four posttraumatic) and 13 associated with ligament lesions ( n = 8) or multiple and severe lesions ( n = 3 indication of salvage). Mean surface of cartilage defects was 6 cm 2. Mean preoperative evolution was 11 months and stage was grade IV (Outerbridge) for all. The first step was arthroscopy for classification and biopsy. The second one was implantation after a 3-week delay (for the ex vivo culture) through arthrotomy, under a periosteal flap taken from tibia and sutured on the edges of the prepared defect. Weight bearing was allowed after the 6th week; MRI was performed at 6, 12, 18, 24 months. The follow up was evaluated with three scales: Lysholm 2, Tegner Activity, Cincinnati Knee Rating System. Results Postoperative complications included: algodystrophy ( n = 2) and phlebitis ( n = 1). Four patients were revised at 6 months, seven between 6 and 12 months, 11 after. The longest follow-up was 26 months. Results were poor in one patient (salvage). For the others, pain and swelling decreased after 6 months and disappeared after 12 months. Conclusion Autologous chondrocyte implantation used in this series and in a large international ongoing series seems to be the only procedure allowing a true long-term regeneration of cartilage defects. Some questions remain, on the biological level in relation with the use of some growth factors and the risk of chromosomic abnormalities, and on the economical level because of the high cost of this technique.

The Manipulated Mesenchymal Stem Cells in Regenerated Skeletal Tissues

Ample experimental examples have been accumulated during the last 3 decades indicating the ability of exogenous sources of cultured cells to serve as implants accelerating cartilage regeneration in defects of articular surfaces. In some cases, the repair tissues form complete spatial reconstruction of the defect. In other cases, either the spatial reconstruction is incomplete or the quality of the reparative tissue is inadequate. A delayed pace of endochondral ossification in the deep zones of the subchondral region of the defects, or ossification above the tide mark, within the superficial cartilaginous articular regions have been noted. Therefore, even in this promising approach of biological resurfacing procedure results are not certain, and further investigative research efforts are required. In the current study, a comparison of implantations of various cultured cells of four different sources were tested in an avian system. The reparative tissue outcomes are divided into three grades: full regeneration success, partial success, and failure of regeneration according to qualitative histological parameters and quantitative observation of the gross specimen. Defects that failed to regenerate a completely filled lesion were found to contain cells carrying the preskeletal–precartilaginous characteristic marker of FGFR3. The findings based on the above parameters suggest that autogeneic, chondrocytic-enriched bone marrow derived mesenchymal cells are superior to other cell sources for articular cartilage regeneration. Grafting of defects with these cells results in a 100% success rate. Allogeneic limb bud-derived mesenchymal cells and allogeneic embryonal chondrocytes have both reached a success of 75% of completely filled defects. Allogeneic chondrocytic-enriched bone marrow-derived mesenchymal cells yielded a 31% success rate. Untreated defects completely failed to heal. In successfully healed defects no cells of the reparative tissue carry the FGFR3 marker 3 months postimplantation. In partially healed defects, FGFR3 positive staining is present in fibrous cells at the invaginated surface. These latest findings may suggest some kind of proliferation failure in such cases.

The manipulated mesenchymal stem cells in regenerated skeletal tissues.

Ample experimental examples have been accumulated during the last 3 decades indicating the ability of exogenous sources of cultured cells to serve as implants accelerating cartilage regeneration in defects of articular surfaces. In some cases, the repair tissues form complete spatial reconstruction of the defect. In other cases, either the spatial reconstruction is incomplete or the quality of the reparative tissue is inadequate. A delayed pace of endochondral ossification in the deep zones of the subchondral region of the defects, or ossification above the tide mark, within the superficial cartilaginous articular regions have been noted. Therefore, even in this promising approach of biological resurfacing procedure results are not certain, and further investigative research efforts are required. In the current study, a comparison of implantations of various cultured cells of four different sources were tested in an avian system. The reparative tissue outcomes are divided into three grades: full regeneration success, partial success, and failure of regeneration according to qualitative histological parameters and quantitative observation of the gross specimen. Defects that failed to regenerate a completely filled lesion were found to contain cells carrying the preskeletal-precartilaginous characteristic marker of FGFR3. The findings based on the above parameters suggest that autogeneic, chondrocytic-enriched bone marrow derived mesenchymal cells are superior to other cell sources for articular cartilage regeneration. Grafting of defects with these cells results in a 100% success rate. Allogeneic limb bud-derived mesenchymal cells and allogeneic embryonal chondrocytes have both reached a success of 75% of completely filled defects. Allogeneic chondrocytic-enriched bone marrow-derived mesenchymal cells yielded a 31% success rate. Untreated defects completely failed to heal. In successfully healed defects no cells of the reparative tissue carry the FGFR3 marker 3 months postimplantation. In partially healed defects, FGFR3 positive staining is present in fibrous cells at the invaginated surface. These latest findings may suggest some kind of proliferation failure in such cases.