Christian Kaps

Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results

Autologous chondrocyte implantation (ACI) is an effective clinical procedure for the regeneration of articular cartilage defects. BioSeed®-C is a second-generation ACI tissue engineering cartilage graft that is based on autologous chondrocytes embedded in a three-dimensional bioresorbable two-component gel-polymer scaffold. In the present prospective study, we evaluated the short-term to mid-term efficacy of BioSeed-C for the arthrotomic and arthroscopic treatment of posttraumatic and degenerative cartilage defects in a group of patients suffering from chronic posttraumatic and/or degenerative cartilage lesions of the knee. Clinical outcome was assessed in 40 patients with a 2-year clinical follow-up before implantation and at 3, 6, 12, and 24 months after implantation by using the modified Cincinnati Knee Rating System, the Lysholm score, the Knee injury and Osteoarthritis Outcome Score, and the current health assessment form (SF-36) of the International Knee Documentation Committee, as well as histological analysis of second-look biopsies. Significant improvement (p < 0.05) in the evaluated scores was observed at 1 and/or 2 years after implantation of BioSeed-C, and histological staining of the biopsies showed good integration of the graft and formation of a cartilaginous repair tissue. The Knee injury and Osteoarthritis Outcome Score showed significant improvement in the subclasses pain, other symptoms, and knee-related quality of life 2 years after implantation of BioSeed-C in focal osteoarthritic defects. The results suggest that implanting BioSeed-C is an effective treatment option for the regeneration of posttraumatic and/or osteoarthritic defects of the knee.

Treatment of focal degenerative cartilage defects with polymer-based autologous chondrocyte grafts: four-year clinical results

IntroductionSecond-generation autologous chondrocyte implantation with scaffolds stabilizing the grafts is a clinically effective procedure for cartilage repair. In this ongoing prospective observational case report study, we evaluated the effectiveness of BioSeed®-C, a cell-based cartilage graft based on autologous chondrocytes embedded in fibrin and a stable resorbable polymer scaffold, for the treatment of clinical symptomatic focal degenerative defects of the knee.MethodsClinical outcome after 4-year clinical follow-up was assessed in 19 patients with preoperatively radiologically confirmed osteoarthritis and a Kellgren-Lawrence score of 2 or more. Clinical scoring was performed before implantation of the graft and 6, 12, and 48 months after implantation using the Lysholm score, the Knee injury and Osteoarthritis Outcome Score (KOOS), the International Knee Documentation Committee (IKDC) score, and the International Cartilage Repair Society (ICRS) score. Cartilage regeneration and articular resurfacing were assessed by magnetic resonance imaging (MRI) 4 years after implantation of the autologous cartilage graft.ResultsSignificant improvement (P < 0.05) of the Lysholm and ICRS scores was observed as early as 6 months after implantation of BioSeed®-C and remained stable during follow-up. The IKDC score showed significant improvement compared with the preoperative situation at 12 and 48 months (P < 0.05). The KOOS showed significant improvement in the subclasses pain, activities of daily living, and knee-related quality of life 6 months as well as 1 and 4 years after implantation of BioSeed®-C in osteoarthritic defects (P < 0.05). MRI analysis showed moderate to complete defect filling with a normal to incidentally hyperintense signal in 16 out of 19 patients treated with BioSeed®-C. Two patients without improvement in the clinical and MRI scores received a total knee endoprosthesis after 4 years.ConclusionsThe results show that the good clinical outcome achieved 1 year after implantation of BioSeed®-C remains stable over the course of a period of 4 years and suggest that implanting BioSeed®-C is a promising treatment option for the repair of focal degenerative defects of the knee.

Human Platelet-Rich Plasma Stimulates Migration and Chondrogenic Differentiation of Human Subchondral Progenitor Cells

In cartilage repair, platelet‐rich plasma (PRP) is used in one‐step approaches utilizing microfracture and matrix‐induced chondrogenesis procedures, bone marrow‐derived cell transplantation, or intra‐articular injection. The aim of our study was to evaluate the effect of human PRP on the migration and chondrogenic differentiation of human subchondral progenitors. Human progenitors were derived from subchondral cortico‐spongious bone (CSP), were analyzed for their migration capacity upon PRP treatment in 96‐well chemotaxis assays and cultured in high‐density pellet cultures under serum‐free conditions in the presence of 5% PRP. Chemotaxis assays showed that 0.1–100% PRP significantly (pu2009<u20090.05) stimulate the migration of CSP compared to untreated controls. Histological staining of proteoglycan and immuno‐staining of type II collagen indicated that progenitors stimulated with PRP show significantly increased cartilage matrix formation compared to untreated progenitors. Real‐time gene expression analysis of typical chondrocyte marker genes as well as osteogenic and adipogenic markers like osteocalcin and fatty acid binding protein showed that PRP induces the chondrogenic differentiation sequence of human progenitors in high‐density pellet cultures, while osteogenic or adipogenic differentiation was not evident. These results suggest that human PRP may enhance the migration and stimulate the chondrogenic differentiation of human subchondral progenitor cells known from microfracture.

Chemokine profile of synovial fluid from normal, osteoarthritis and rheumatoid arthritis patients: CCL25, CXCL10 and XCL1 recruit human subchondral mesenchymal progenitor cells

OBJECTIVEnThe microfracture technique activates mesenchymal progenitors that enter the cartilage defect and form cartilage repair tissue. Synovial fluid (SF) has been shown to stimulate the migration of subchondral progenitors. The aim of our study was to determine the chemokine profile of SF from normal, rheumatoid arthritis (RA) and osteoarthritis (OA) donors and evaluate the chemotactic effect of selected chemokines on human subchondral progenitor cells.nnnMETHODnChemokine levels of SF were analyzed using human chemokine antibody membrane arrays. The chemotactic potential of selected chemokines on human mesenchymal progenitors derived from subchondral cortico-spongious bone was tested using 96-well chemotaxis assays. Chemokine receptor expression of subchondral progenitors was assessed by real-time gene expression analysis and immuno-histochemistry.nnnRESULTSnChemokine antibody array analysis showed that SF contains a broad range of chemokines. Ten chemokines that showed significantly reduced levels in RA or OA compared to normal SF or robustly high levels in all SF tested were used for further chemotactic analysis. Chemotaxis assays showed that the chemokines MDC/CCL22, CTACK/CCL27, ENA78/CXCL5 and SDF1α/CXCL12 significantly inhibited migration of progenitors, while TECK/CCL25, IP10/CXCL10 and Lymphotactin/XCL1 effectively stimulated cell migration. MCP1/CCL2, Eotaxin2/CCL24 and NAP2/CXCL7 showed no chemotactic effect on subchondral progenitors. Gene expression and immuno-histochemical analysis of corresponding chemokine receptors document presence of low levels of chemokine receptors in subchondral progenitors, with the CXCL10 receptor CXCR3 showing the highest expression level.nnnCONCLUSIONnThese results suggest that SF contains chemokines that may contribute to the recruitment of human mesenchymal progenitors from the subchondral bone in microfracture.

Chondrogenic differentiation capacity of human mesenchymal progenitor cells derived from subchondral cortico-spongious bone.

Microfracture is frequently used to repair articular cartilage defects and allows mesenchymal progenitors to migrate from subchondral bone into the defect and form cartilaginous repair tissue. The aim of our study was to analyze the cell surface antigen pattern and the differentiation capacity of cells derived from human subchondral bone. Human progenitor cells were derived from subchondral cortico‐spongious bone and grown in the presence of human serum. Stem cell‐related cell surface antigens were analyzed by flowcytometry. Cortico‐spongious progenitor (CSP) cells showed presence of CD73, CD90, CD105, and STRO‐1. Multilineage differentiation potential of CSP cells was documented by histological staining and by gene expression analysis of osteogenic, adipogenic, and chondrogenic marker genes. CSP cells formed a mineralized matrix as demonstrated by von Kossa staining and showed induction of osteocalcin, independent of osteogenic stimulation. During adipogenic differentiation, the adipogenic marker genes fatty acid binding protein 4 and peroxisome proliferative activated receptor γ were induced. Immunohistochemical staining of cartilage‐specific type II collagen and induction of the chondrocytic marker genes cartilage oligomeric matrix protein, aggrecan, and types II and IX collagen confirmed TGFβ3‐mediated chondrogenic lineage development. CSP cells from subchondral bone, as known from microfracture, are multipotent stem cell‐like mesenchymal progenitors with a high chondrogenic differentiation potential.

Human mastoid periosteum‐derived stem cells: promising candidates for skeletal tissue engineering

Currently, mesenchymal stem cells (MSCs) are considered as the most eligible cells for skeletal tissue engineering. However, factors such as difficult stimulation and control of differentiation in vivo hamper their clinical use. In contrast, periosteum or periosteum‐derived cells (PCs) are routinely clinically applied for bone and cartilage repair. PCs have often been named MSCs but, although cells of osteochondrogenic lineages arise from MSCs, it is unclear whether periosteum really contains MSCs. Our aim was to investigate the MSC‐like character of PCs derived from the periosteum of mastoid bone. Harvesting of periosteum from mastoid bone is easy, so mastoid represents a good source for the isolation of PCs. Therefore, we analysed the MSC‐like growth behaviour and the expression of embryonic, ectodermal, endodermal and mesodermal markers by microarray and FACS technology, and the multilineage developmental capacity of human PCs. Regarding clinical relevance, experiments were performed in human serum‐supplemented medium. We show that PCs do not express early embryonic stem cell markers such as Oct4 and Nanog, or the marker of haematopoietic stem cells CD34, but express some other MSC markers. Osteogenesis resulted in the formation of calcified matrix, increased alkaline phosphatase activity, and induction of the osteogenic marker gene osteocalcin. Staining of proteoglycans and deposition of type II collagen documented chondrogenic development. As shown for the first time, adipogenic stimulation of mastoid‐derived PCs resulted in the formation of lipid droplets and expression of the adipogenic marker genes aP2 and APM1. These results suggest MSC‐like PCs from mastoid as candidates for therapy of complex skeletal defects. Copyright

Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1β, TNF-α, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology.

The reprogrammed host: Chlamydia trachomatis–induced up-regulation of glycoprotein 130 cytokines, transcription factors, and antiapoptotic genes

OBJECTIVEnInfection with Chlamydia trachomatis is a known cause of sexually transmitted diseases, eye infections (including trachoma), and reactive arthritis (ReA). Because the mechanisms of Chlamydia-induced changes leading to ReA are poorly defined, this study sought to identify the target genes involved at the molecular level.nnnMETHODSnChlamydia-induced changes in host cells were investigated by combining a screening technique, which utilized complementary DNA arrays on C trachomatis-infected and mock-infected epithelial HeLa cells, with real-time reverse transcription-polymerase chain reaction or enzyme-linked immunosorbent assay of gene products. Some responses were additionally demonstrated on human primary chondrocytes and a human synovial fibroblast cell line, both of which served as model cells for ReA.nnnRESULTSnEighteen genes (of 1,176) were found to be up-regulated after 24 hours of infection with this obligate intracellular bacterium, among them the glycoprotein 130 family members IL-11 and LIF, the chemokine gene MIP2-alpha, the transcription factor genes EGR1, ETR101, FRA1, and c-jun, the apoptosis-related genes IEX-1L and MCL-1, adhesion molecule genes such as ICAM1, and various other functionally important genes. In the context of this rheumatic disease, the cytokines and transcription factors seem to be especially involved, since various connections to chondrocytes, synoviocytes, bone remodeling, joint pathology, and other rheumatic diseases have been demonstrated.nnnCONCLUSIONnInfection with C trachomatis seems to reprogram the host cells (independent of activation by lipopolysaccharide or other ultraviolet-resistant bacterial components) at various key positions that act as intra- or intercellular switches, suggesting that these changes and similar Chlamydia-induced functional alterations constitute an important basis of the pathogenic inflammatory potential of these cells in ReA. Our results suggest that this approach is generally useful for the broad analysis of host-pathogen interactions involving obligate intracellular bacteria, and for the identification of target genes for therapeutic intervention in this rheumatic disease.

Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: implications for tendon tissue engineering.

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue.

BMP7 promotes adipogenic but not osteo‐/chondrogenic differentiation of adult human bone marrow‐derived stem cells in high‐density micro‐mass culture

The objective of our study was to elucidate the potential of bone morphogenetic protein‐7 (BMP7) to initiate distinct mesenchymal lineage development of human adult mesenchymal stem cells (MSC) in three‐dimensional micro‐mass culture. Expanded MSC were cultured in high‐density micro‐masses under serum‐free conditions that favor chondrogenic differentiation and were stimulated with 50–200 ng/ml BMP7 or 10 ng/ml transforming growth factor‐β3 (TGFβ3) as control. Histological staining of proteoglycan with alcian blue, mineralized matrix according to von Kossa, and lipids with Oil Red O, immunostaining of type II collagen as well as real‐time gene expression analysis of typical chondrogenic, adipogenic, and osteogenic marker genes showed that BMP7 promoted adipogenic differentiation of MSC. Micro‐masses stimulated with BMP7 developed adipocytic cells filled with lipid droplets and showed an enhanced expression of the adipocyte marker genes fatty acid binding protein 4 (FABP4) and the adipose most abundant transcript 1 (apM1). Development along the chondrogenic lineage or stimulation of osteogenic differentiation were not evident upon stimulation with BMP7 in different concentrations. In contrast, TGFβ3 directed MSC to form a cartilaginous matrix that is rich in proteoglycan and type II collagen. Gene expression analysis of typical chondrocyte marker genes like cartilage oligomeric matrix protein (COMP), link protein, aggrecan, and types IIα1 and IXα3 collagen confirmed chondrogenic differentiation of MSC treated with TGFβ3. These results suggest that BMP7 promotes the adipogenic and not the osteogenic or chondrogenic lineage development of human stem cells when assembled three‐dimensionally in micro‐masses. J. Cell. Biochem. 102: 626–637, 2007.