Increased Chondroprotective Effect of Combining Hyaluronic Acid with a Glucocorticoid Compared to Separate Administration on Cytokine-Treated Osteoarthritic Chondrocytes in a 2D Culture.

Intra-articular injections of glucocorticoids (GC) or hyaluronic acid (HA) are commonly used interventions for patients suffering from knee osteoarthritis (OA). Both substances are combined to achieve a chondroprotective and anti-inflammatory effect. Clinical studies have shown benefits, but data on the cellular level are still lacking. This study aimed to investigate the effect of the GC triamcinolone hexacetonide, HA, and a mix of both substances on cytokine-treated chondrocytes in vitro. Chondrocytes isolated from human articular cartilage were seeded on 6- and 24-well plates. Mimicking OA's inflammatory state, cells were treated with IL-1ß and IL-17 for six days, whereby, after three days, test substances (10%) were added to the culture medium. Chondrocytes were analyzed on days three and six concerning their actin polymerization, expression of anabolic and catabolic genes, metabolic activity, cytokine release, and reactive oxygen species (ROS). Adding HA or GC/HA to the inflammatory culture medium increased the metabolic activity of chondrocytes, while groups containing GC reduced catabolic gene expression and the release of TNF-α. In addition, enhanced F-actin content was shown supplementing HA or GC/HA to the culture medium. Supplementing GC with HA leads to an anti-inflammatory and chondroprotective effect by diminishing the side effects of GC supplementation alone.

Hyaluronic Acid as a Carrier Supports the Effects of Glucocorticoids and Diminishes the Cytotoxic Effects of Local Anesthetics in Human Articular Chondrocytes In Vitro.

The current study aimed to investigate the cytotoxicity of co-administrating local anesthetics (LA) with glucocorticoids (GC) and hyaluronic acid (HA) in vitro. Human articular cartilage was obtained from five patients undergoing total knee arthroplasty. Chondrocytes were isolated, expanded, and seeded in 24-well plates for experimental testing. LA (lidocaine, bupivacaine, ropivacaine) were administered separately and co-administered with the following substances: GC, HA, and GC/HA. Viability was confirmed by microscopic images, flow cytometry, metabolic activity, and live/dead assay. The addition of HA and GC/HA resulted in enhanced attachment and branched appearance of the chondrocytes compared to LA and LA/GC. Metabolic activity was better in all LA co-administered with HA and GC/HA than with GC and only LA. Flow cytometry revealed the lowest cell viability in lidocaine and the highest cell viability in ropivacaine. This finding was also confirmed by live/dead assay. In conclusion, HA supports the effect of GC and reduces chondrotoxic effects of LA in vitro. Thereby, the co-administration of HA to LA and GC offers an alternative less chondrotoxic approach for treating patients with symptomatic osteoarthritis of the knee.

Biotribological Tests of Osteochondral Grafts after Treatment with Pro-Inflammatory Cytokines.

OBJECTIVE: During osteoarthritis progression, cartilage degrades in a manner that influences its biomechanical and biotribological properties, while chondrocytes reduce the synthesis of extracellular matrix components and become apoptotic. This study investigates the effects of inflammation on cartilage under biomechanical stress using biotribological tests. METHODS: Bovine osteochondral grafts from five animals were punched out from the medial condyle and treated with or without pro-inflammatory cytokines (interleukin-1ß [IL-1ß], tumor necrosis factor-α [TNF-α], IL-6) for 2 weeks. After incubation, biotribological tests were performed for 2 hours (alternating 10 minutes test and pause respectively at 39°C, 180 N, 1 Hz, and 2 mm stroke). Before and after testing, the cartilage surface was imaged with a 3-dimensional microscope. During testing, the coefficient of friction (COF) was measured, while gene expression analysis and investigation of metabolic activity of chondrocytes were carried out after testing. Histological sections of the tissue and wear debris from the test fluid were also analyzed. RESULTS: After biotribological tests, surface cracks were found in both treated and untreated osteochondral grafts. In treated grafts, the COF increased, and the proteoglycan content in the cartilage tissue decreased, leading to structural changes. Chondrocytes from treated grafts showed increased expression of genes encoding for degradative enzymes, while cartilage-specific gene expression and metabolic activity exhibited no significant differences between treated and untreated groups. No measurable difference in the wear debris in the test fluid was found. CONCLUSIONS: Treatment of osteochondral grafts with cytokines results in a significantly increased COF, while also leading to significant changes in cartilage proteoglycan content and cartilage matrix compression during biotribological tests.

Concentration-Dependent Effects of Cobalt and Chromium Ions on Osteoarthritic Chondrocytes.

OBJECTIVE: Cobalt and chromium (CoCr) ions from metal implants are released into the joint due to biotribocorrosion, inducing apoptosis and altering gene expression in various cell types. Here, we asked whether CoCr ions concentration-dependently changed viability, transcriptional activity, and inflammatory response in human articular chondrocytes. DESIGN: Human articular chondrocytes were exposed to Co (1.02-16.33 ppm) and Cr (0.42-6.66 ppm) ions and cell viability and early/late apoptosis (annexin V and 7-AAD) were assessed in 2-dimensional cell cultures using the XTT assay and flow cytometry, respectively. Changes in chondrocyte morphology were assessed using transmitted light microscopy. The effects of CoCr ions on transcriptional activity of chondrocytes were evaluated by quantitative polymerase chain reaction (qPCR). The inflammatory responses were determined by measuring the levels of released pro-inflammatory cytokines (interleukin-1ß [IL-1ß], IL-6, IL-8, and tumor necrosis factor-α [TNF-α]). RESULTS: CoCr ions concentration-dependently reduced metabolic activity and induced early and late apoptosis after 24 hours in culture. After 72 hours, the majority of chondrocytes (>90%) were apoptotic at the highest concentrations of CoCr ions (16.33/6/66 ppm). SOX9 expression was concentration-dependently enhanced, whereas expression of COL2A1 linearly decreased after 24 hours. IL-8 release was enhanced proportionally to CoCr ions levels, whereas IL-1ß, IL-6, and TNF-α levels were not affected by the treatments. CONCLUSIONS: CoCr ions showed concentration- and time-dependent effects on articular chondrocytes. Fractions of apoptotic articular chondrocytes were proportional to CoCr ion concentrations. In addition, metabolic activity and expression of chondrocyte-specific genes were decreased by CoCr ions. Furthermore, exposure to CoCr ions caused a release of pro-inflammatory cytokines.

Serum albumin-coated bone allograft (BoneAlbumin) results in faster bone formation and mechanically stronger bone in aging rats.

Serum albumin-coated bone allografts (BoneAlbumin) have successfully supported bone regeneration in various experimental models by activating endogenous progenitors. However, the effect of tissue aging, linked to declining stem cell function, has yet to be explicitly examined within the context of BoneAlbumin's regenerative capacity. Stem cell function was tested with an in vitro attachment assay, which showed that albumin coating increases stem cell attachment on demineralized bone surfaces in an aging cell population. Bone regeneration was investigated in vivo by creating critical size bone defects on the parietal bones of aging female rats. Demineralized bone matrices with and without serum albumin coating were used to fill the defects. Bone regeneration was determined by measuring the density and the size of the remaining bone defect with computed tomography (CT). Microcomputed tomography (MicroCT) and mechanical testing were performed on the parietal bone explants. In vivo CT and ex vivo microCT measurements showed better regeneration with albumin-coated grafts. Additionally, the albumin-coated group showed a twofold increase in peak fracture force compared with uncoated allografts. In the present study, serum albumin-coated demineralized bone matrices successfully supported faster and functionally superior bone regeneration in aging rats. Because stem cell function, a key contributor of bone remodelling, decreases with age and serum albumin is an effective activator of endogenous progenitor cells, this method could be an effective and safe adjuvant in bone regeneration of aging adult and osteo-compromised populations.

Effect of osteochondral graft orientation in a biotribological test system.

Autologous osteochondral transplantation (AOT) utilizing autografts is a widely used technique for the treatment of small-to-medium cartilage defects occurring in knee and ankle joints. The application of viable cartilage and bone ensures proper integration, early weight bearing, as well as restoration of biomechanical and biotribological properties. However, alignment of the autografts onto the defect site remains a pivotal aspect of reinstating the properties of the joint toward successful autograft integration. This is the first study to perform tests with different orientations of osteochondral grafts in a cartilage-on-cartilage test system. The objective was to estimate if there are differences between aligned and 90°-rotated grafts concerning molecular biological and biomechanical parameters. Tissue viability, assessed by XTT assay indicated lower metabolic activity in tested osteochondral grafts (aligned, p = 0.0148 and 90°-rotated, p = 0.0760) in favor of a higher anabolic gene expression (aligned, p = 0.0030 and 90°-rotated, 0.0027). Tissue structure was evaluated by Safranin O histology and microscopic images of the surface. Aligned and 90°-rotated grafts revealed no apparent differences between proteoglycan content or cracks and fissures on the cartilage surface. Test medium analyzed after tribological tests for their sulfated glycosaminoglycan content revealed no differences (p = 0.3282). During the tests, both the friction coefficient and the relative displacement between the two cartilage surfaces were measured, with no significant difference in both parameters (COF, p = 0.2232 and relative displacement, p = 0.3185). From the methods we deployed, this study can infer that there are no differences between aligned and 90°-rotated osteochondral grafts after tribological tests in the used ex vivo tissue model. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res.

Redifferentiation of Articular Chondrocytes by Hyperacute Serum and Platelet Rich Plasma in Collagen Type I Hydrogels.

Matrix-assisted autologous chondrocyte transplantation (MACT) for focal articular cartilage defects often fails to produce adequate cartilage-specific extracellular matrix in vitro and upon transplantation results in fibrocartilage due to dedifferentiation during cell expansion. This study aimed to redifferentiate the chondrocytes through supplementation of blood-products, such as hyperacute serum (HAS) and platelet-rich plasma (PRP) in vitro. Dedifferentiated monolayer chondrocytes embedded onto collagen type I hydrogels were redifferentiated through supplementation of 10% HAS or 10% PRP for 14 days in vitro under normoxia (20% O2) and hypoxia (4% O2). Cell proliferation was increased by supplementing HAS for 14 days (p < 0.05) or by interchanging from HAS to PRP during Days 7⁻14 (p < 0.05). Sulfated glycosaminoglycan (sGAG) content was deposited under both HAS, and PRP for 14 days and an interchange during Days 7⁻14 depleted the sGAG content to a certain extent. PRP enhanced the gene expression of anabolic markers COL2A1 and SOX9 (p < 0.05), whereas HAS enhanced COL1A1 production. An interchange led to reduction of COL1A1 and COL2A1 expression marked by increased MMP13 expression (p < 0.05). Chondrocytes secreted less IL-6 and more PDGF-BB under PRP for 14 days (p < 0.0.5). Hypoxia enhanced TGF-ß1 and BMP-2 release in both HAS and PRP. Our study demonstrates a new approach for chondrocyte redifferentiation.

Platelet-Rich Plasma Supports Proliferation and Redifferentiation of Chondrocytes during In Vitro Expansion.

Articular cartilage regeneration is insufficient to restore sports injuries or defects that can occur from trauma. Treatment options for cartilage repair include autologous chondrocyte implantation (ACI) by isolation, expansion, and reimplantation of healthy donor chondrocytes. Chondrocyte expansion onto 2D substrates leads to dedifferentiation and loss of the cellular phenotype. We aimed to overcome the state of dedifferentiation by biochemical stimuli with platelet derivatives such as platelet-rich plasma (PRP) and hyperacute serum (HAS) to achieve sufficient cell numbers in combination with variable oxygen tension. Human articular chondrocytes from osteoarthritic (OA) cartilage chondrocytes were switched from 10% FCS supplementation to either 10% PRP or 10% HAS after initial passaging for further experiments under normoxic (20% O2) or hypoxic (1% O2) conditions. An XTT assay measured the effect of PRP or HAS on the cell proliferation at 3, 6, and 9 days. The chondrogenic redifferentiation potential of dedifferentiated chondrocytes was determined with reverse transcriptase quantitative real-time PCR for markers of expression for type II collagen (COL2A1), type I collagen (COL1A1), and matrix metalloproteinases MMP3, matrix metalloproteinase 13 (MMP13) at 24 and 72 h. Measured protein levels of 100% PRP or HAS by multiplex quantification revealed basic fibroblast growth factor, G-CSF, and PDGF were significantly higher in PRP than in HAS (p < 0.05) but LEPTIN levels did not differ. The quantified protein levels did not differ when isolated from same donors at a different time. Chondrocyte proliferation indicated that supplementation of 10% HAS enhanced the proliferation rate compared to 10% PRP or 10% FCS at 6 and 9 days significantly (p < 0.05). mRNA levels for expression of COL1A1 were significantly downregulated (p < 0.05) when cultured with 10% PRP than 10% HAS or 10% FCS under normoxic/hypoxic conditions. COL2A1 was significantly upregulated (p < 0.05) in PRP than 10% HAS or 10% FCS. MMP3 expression was downregulated after 72 h under all conditions. MMP13 was upregulated with 10% PRP at both 24 and 72 h but significantly downregulated under hypoxia (1% O2) for all circumstances. While HAS has its effect on chondrocyte proliferation, PRP enhances both proliferation and redifferentiation of dedifferentiated chondrocytes. PRP can replace standard usage of FCS for chondrogenic priming and expansion as implications for clinical use such as ACI procedures.

A protocol for gene expression analysis of chondrocytes from bovine osteochondral plugs used for biotribological applications.

RNA isolation from human or animal cartilage tissue is necessary when performing mechanical or biotribological applications. Despite no influence on the cells and no alterations in gene expression patterns, enzymatic digestion of tissues should be avoided as it's known that the expression of collagen 2 can be effected (Hayman et al., 2006 [1]). After mechanical or biotribological tests alternative options with an immediate disruption of the tissue should be contemplated. To obtain RNA, different tissue homogenization and disruption methods are available on the market (Yu et al., 2004 [2]), but not everyone is suitable for cartilage. Some of them neither homogenize the cartilage, while others are producing a lot of foam during disruption process. After trying some of the currently available methods, we chose the MagNA Lyser Instrument from Roche to disrupt the cartilage and further isolate RNA by using the Fibrous Tissue Kit from Qiagen. After RNA isolation, cDNA synthesis was performed by additionally adding RNA from bacteriophage MS2 for stabilization purposes. For the RTqPCR bovine primers were designed and tested for efficiency to confirm that the whole gene expression analysis is working. Our protocol explains a whole method to perform gene expression analysis from bovine cartilage, but can also be used for human or any other animal tissue.

Hyaluronan thiomer gel/matrix mediated healing of articular cartilage defects in New Zealand White rabbits-a pilot study.

BACKGROUND: Articular cartilage defects are limited to their regenerative potential in human adults. Our current study evaluates tissue regeneration in a surgically induced empty defect site with hyaluronan thiomer as a provisional scaffold in a gel/matrix combination without cells on rabbit models to restore tissue formation. METHODS: An osteochondral defect of 4 mm in diameter and 5 mm in depth was induced by mechanical drilling in the femoral center of the trochlea in 18 New Zealand White rabbits. Previously evaluated from an in vitro study hyaluronan thiomer matrix, and a hyaluronan thiomer gel was used to treat the defect. As a control, the defect was left untreated. During the whole study, rabbits were clinically examined and after 4 (n = 3) or 12 (n = 3) weeks, the rabbits were sacrificed. Joints were evaluated macroscopically (Brittberg score) and by histology (O'Driscoll score). Synovial cells from the synovial fluid smear were histopathologically evaluated. RESULTS: The healing of the defects varied intra-group wise at the first observation period. After 12 weeks the results concerning the cartilage repair score were inhomogeneous within each group, while the macroscopic analysis was more homogenous. In the synovial fluid smear, the mean score of infiltrated synovial and non-synovial cells was slightly increased after 4 weeks and slightly decreased after 12 weeks in both the treatment groups in comparison to the untreated control. CONCLUSIONS: Taken together with results from the in vivo study indicated that implantation of hyaluronan thiomer as a combination of gel and matrix might enhance articular cartilage regeneration in an empty defect. Despite their benefits, the intrinsic healing capacity of New Zealand rabbits is a limitation for comparative test subject in pre-clinical models of cartilage defects.

Chondrogenic Gene Expression Differences between Chondrocytes from Osteoarthritic and Non-OA Trauma Joints in a 3D Collagen Type I Hydrogel.

Objective The purpose of the current study was to compare the donor age variation of chondrocytes from non-OA (osteoarthritic) trauma joints in patients of young to middle age (20.5 ± 3.7, 31.8 ± 1.9, 41.9 ± 4.1 years) embedded in matrix-associated autologous chondrocyte transplantation (MACT) grafts (CaReS). The chondrocyte-specific gene expression of CaReS grafts were then compared to chondrocytes from OA joints (in patients aged 63.8 ± 10 years) embedded in a collagen type I hydrogel. Design OA chondrocytes and articular chondrocyte-laden grafts were cultured over 14 days in chondrogenic growth medium. We performed reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate the mRNA expression levels of chondrocyte-specific and hypertrophic markers. Results Gene expression analysis with RT-qPCR revealed no significant difference in chondrocyte-specific genes ( COL2A1, ACAN, SOX9, SOX5, SOX6) among 3 different age group of patients with CaReS grafts. In a comparative analysis of OA chondrocytes to articular chondrocytes, chondrogenic markers ( COL2A1, SOX6) exhibited higher expression in OA chondrocytes ( P < 0.05). Hypertrophic or OA cartilage pathogenesis marker ( MMP3, MMP13) expression was higher and COL1A1 had significantly lower expression ( P < 0.05) in OA chondrocytes than articular chondrocytes when cultivated in collagen type I hydrogels. Conclusion In summary, we identify that donor age variation does not influence the chondrogenic gene expression of the CaReS system. We also identified that freshly isolated OA chondrocytes embedded in collagen type I hydrogels can exhibit chondrogenic gene expression as observed in articular chondrocytes on the CaReS grafts. Transforming OA chondrocytes to articular chondrocytes can be regarded as an alternative option in the MACT technique.

Chondroprotective effect of high-molecular-weight hyaluronic acid on osteoarthritic chondrocytes in a co-cultivation inflammation model with M1 macrophages.

BACKGROUND: Osteoarthritis (OA) is described by an imbalance between anabolic and catabolic processes in the affected joint. This dysregulation of metabolism affects not only chondrocytes within cartilage tissue but also the cells of the synovial membrane across the border of the joint. An important factor in OA is the low viscosity of the synovial fluid. High-molecular-weight hyaluronic acid (HA) can be used to increase the viscosity and also reduce inflammatory processes. The purpose was to establish an in vitro inflammation model and to evaluate the effects of high-molecular-weight HA in a co-cultivation inflammation model of osteoarthritic chondrocytes and M1 macrophages. METHODS: For the establishment of the inflammation model THP-1 cells were, at first, differentiated to M0 macrophages and then activated to the M1 subtype after 5 days of resting period. Surface markers, cytokine release, and gene expression, were analyzed to examine the successful differentiation. In the inflammation model, the defined M1 macrophages were co-cultivated with osteoarthritic chondrocytes for 2 days, with and without the addition of 10 % HA and further analyzed for chondrogenic gene expression markers and the release of cytokines in the supernatant. RESULTS: The differentiation and activation process was successful as M1 macrophages expressed higher levels of pro-inflammatory cytokines and specific genes. Similarly, the surface marker CD14 was significantly decreased compared to M0 macrophages. For the co-culture system, the analysis of gene expression showed that HA increased the expression of cartilage-specific genes while catabolic-encoding genes exhibited lower expression levels than the control group. This positive effect of HA was also demonstrated by the measurement of pro-inflammatory cytokines, as their level decreased. CONCLUSION: Our study implies that high-molecular-weight HA has a chondroprotective effect in the present co-cultivation inflammation model, as it decreases pro-inflammatory cytokines and increases anabolic factors.

A Novel Cross-Linked Hyaluronic Acid Porous Scaffold for Cartilage Repair: An In Vitro Study With Osteoarthritic Chondrocytes.

PURPOSE: An important feature of biomaterials used in cartilage regeneration is their influence on the establishment and stabilization of a chondrocytic phenotype of embedded cells. The purpose of this study was to examine the effects of a porous 3-dimensional scaffold made of cross-linked hyaluronic acid on the expression and synthesis performance of human articular chondrocytes. MATERIALS AND METHODS: Osteoarthritic chondrocytes from 5 patients with a mean age of 74 years were passaged twice and cultured within the cross-linked hyaluronic acid scaffolds for 2 weeks. Analyses were performed at 3 different time points. For estimation of cell content within the scaffold, DNA-content (CyQuant cell proliferation assay) was determined. The expression of chondrocyte-specific genes by embedded cells as well as the total amount of sulfated glycosaminoglycans produced during the culture period was analyzed in order to characterize the synthesis performance and differentiation status of the cells. RESULTS: Cells showed a homogenous distribution within the scaffold. DNA quantification revealed a reduction of the cell number. This might be attributed to loss of cells from the scaffold during media exchange connected with a stop in cell proliferation. Indeed, the expression of cartilage-specific genes and the production of sulfated glycosaminoglycans were increased and the differentiation index was clearly improved. CONCLUSIONS: These results suggest that the attachment of osteoarthritic P2 chondrocytes to the investigated material enhanced the chondrogenic phenotype as well as promoted the retention.

Correlation Analysis of SOX9, -5, and -6 as well as COL2A1 and Aggrecan Gene Expression of Collagen I Implant-Derived and Osteoarthritic Chondrocytes.

OBJECTIVE: Matrix-assisted autologous chondrocyte implantation is frequently applied to replace damaged cartilage in order to support tissue regeneration or repair and to prevent progressive cartilage degradation and osteoarthritis. Its application, however, is limited to primary defects and contraindicated in the case of osteoarthritis that is partially ascribed to dedifferentiation and phenotype alterations of chondrocytes obtainable from patients' biopsies. The differentiation state of chondrocytes is reflected at the level of structural gene (COL2A1, ACAN, COL1A1) and transcription factor (SOX9, 5, 6) expression. METHODS/DESIGN: We determined the mRNA abundances of COL2A1, ACAN, and COL1A1as well as SOX9, -5, and -6 of freshly isolated and passaged collagen I implant-derived and osteoarthritic chondrocytes via reverse transcription-polymerase chain reaction. Moreover, we analyzed the correlation of structural and transcription factor gene expression. Thus, we were able to evaluate the impact of the mRNA levels of transcription factors on the expression of cartilage-specific structural genes. RESULTS: Significant differences were obtained (1) for freshly isolated osteoarthritic versus collagen I implant-derived chondrocytes, (2) due to passaging of the respective cell sources, (3) for osteoarthritic versus nonosteoarthritic chondrocytes, and (4) for COL2A1 versus ACAN expression with respect to the coherence with SOX9, -5, and -6 transcript levels. CONCLUSION: Our results might contribute to a better understanding of the transcriptional regulation of structural gene expression of chondrocytes with implications for their use in matrix-assisted autologous chondrocyte implantation.

Mechanostimulation changes the catabolic phenotype of human dedifferentiated osteoarthritic chondrocytes.

PURPOSE: The treatment of cartilage defects with matrix-embedded autologous chondrocytes is a promising method to support the repair process and to foster reconstitution of full functionality of the joint. METHODS: Human osteoarthritic chondrocytes were harvest from nine different patients (mean ± SD age 68 ± 8 years) who underwent total knee replacement. The chondrocytes were embedded after a precultivation phase into a collagen I hydrogel. Mid-term intermitted mechanostimulation on matrix-embedded dedifferentiated human osteoarthritic chondrocytes was performed by intermittently applying a cyclic sinusoid compression regime for 4 days (cycles of 1 h of sinusoidal stimulation (1 Hz) and 4 h of break; maximum compression 2.5%). Stimulated (Flex) and non-stimulated (No Flex) cell matrix constructs were analysed concerning the expression of genes involved in tissue metabolism, the content of sulphated glycosaminoglycans (sGAG) and the morphology of the chondrocytes. RESULTS: Gene expression analysis showed a high significant increase in collagen type II expression (p < 0.001), a significant increase in aggrecan expression (p < 0.04) and a high significant decrease in MMP-13 expression (p < 0.001) under stimulation condition compared with unstimulated controls. No significant changes were found in the gene expression rate of MMP-3. This positive effect of the mechanostimulation was confirmed by the analyses of sGAG. Mechanically stimulated cell-matrix constructs had nearly tripled sGAG content than the non-stimulated control (p < 0.002). In addition, histological examination showed that morphology of chondrocytes was altered from a spindle-shaped to a chondrocyte-characteristic rounded phenotype. CONCLUSION: Mid-term intermitted mechanical stimulation in vitro has the potential to improve the cell quality of cell matrix constructs prepared from dedifferentiated osteoarthritic chondrocytes. This observation may extend the inclusion criteria for matrix-assisted autologous chondrocyte implantation (MACI) and confirms the importance of moderate dynamic compression in clinical rehabilitation after MACI.

Investigation of bone allografts representing different steps of the bone bank procedure using the CAM-model.

Bone grafting is commonly used to treat large bone defects. Since autografts are limited and frequently associated with postoperative donor morbidity, allografts from bone banks are often used. However, vascularisation of the allograft is often impaired, resulting in inadequate bone healing and functional graft failure. In bone bank processing, tissue is stored at -80 degree Celsius and subsequently subjected to a harsh multi-step cleaning and sterilisation procedure to prevent immune rejection or transmission of diseases. To determine which step of this procedure diminishes the ability of allografts to induce or promote vascularisation, we used the chick chorioallantoic membrane (CAM) model to monitor the vascular reaction to sample bone chips representing the respective procedural steps. The CAM model monitors the angiogenic potency of xenogeneic and, hence, potentially immunogeneic materials (e.g. cells, tissues, tissue-engineered matrices). Due to the chicken embryo's lack of a fully functional immune system, it provides test conditions that are analogous to immunologically incompetent mice and is a well-suited alternative to their use. Bone chips were placed onto the CAM, and vascular reactions were quantified by image analysis after 48 h incubation. The vascular reaction was most pronounced to fresh, untreated bone chips that had been kept at +2 degree Celsius prior to the experiment. Surprisingly, storage of bone samples at -80 degree Celsius was sufficient to drastically reduce the vascular reaction. Consistent with this, samples representing different stages of the subsequent procedure showed similarly low vascular indices.

Investigation of Collagen Transplants Seeded with Human Autologous Chondrocytes at the Time of Transplantation.

OBJECTIVE: The treatment of cartilage defects with matrix-embedded autologous chondrocytes is a promising method to support the repair process. In this study we gathered quality parameters of collagen I matrices and embedded autologous chondrocytes at the time of transplantation We determined number, morphology, and distribution of matrix-embedded chondrocytes as well as their synthesis performance concerning sulphated glycosaminoglycans (sGAG) and collagen 1A1 and 2A1 mRNA levels. RESULTS: Chondrocytes were equidistantly distributed in the collagen matrices, and cell numbers ranged from 6 to 34 × 10(4) cells/g wet weight. Significant amounts of sGAG were detected in all of the investigated transplants but did not correlate with the number of cells within the respective transplants. Moreover, collagen I mRNA levels exceeded that of collagen II up to 17-fold. Collagen I and II ratio and sGAG amounts indicated significant interindividual differences of chondrocytes. The variation of transplant-associated sGAG levels could be attributed to the differential biosynthesis performance of chondrocytes. CONCLUSIONS: These results confirm the vitality and the chondrocytic phenotype of matrix-embedded cells (CaRes(®)) with respect to sGAG synthesis. However, chondrocytes showed collagen I mRNA expression partially far exceeding that of collagen II, indicating a rather dedifferentiated cellular status. In addition, sGAG synthesis performance of different patients' chondrocytes varied significantly. Nevertheless, a 2-year clinical study of chondrocyte-seeded collagen matrices as investigated in this work delivered promising results. However, future studies are planned to determine markers for the regenerative potential of donor chondrocytes.