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1.  Regenerative and Immunogenic Characteristics of Cultured Nucleus Pulposus Cells from Human Cervical Intervertebral Discs 
PLoS ONE  2015;10(5):e0126954.
Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.
PMCID: PMC4438063  PMID: 25993467
2.  N-Glycosylation Profile of Undifferentiated and Adipogenically Differentiated Human Bone Marrow Mesenchymal Stem Cells: Towards a Next Generation of Stem Cell Markers 
Stem Cells and Development  2013;22(23):3100-3113.
Mesenchymal stem cells (MSCs) are multipotent cells that are easy to isolate and expand, develop into several tissues, including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. This makes MSCs ideal for regenerative medicine. For application and regulatory purposes, knowledge of (bio)markers characterizing MSCs and their development stages is of paramount importance. The cell surface is coated with glycans that possess lineage-specific nature, which makes glycans to be promising candidate markers. In the context of soft tissue generation, we aimed to identify glycans that could be markers for MSCs and their adipogenically differentiated progeny. MSCs were isolated from human bone marrow, adipogenically stimulated for 15 days and adipogenesis was verified by staining the lipid droplets and quantitative real time polymerase chain reaction of the marker genes peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid binding protein-4 (FABP4). Using matrix-assisted laser desorption-ionization-time of flight mass spectrometry combined with exoglycosidase digestions, we report for the first time the N-glycome of MSCs during adipogenic differentiation. We were able to detect more than 100 different N-glycans, including high-mannose, hybrid, and complex N-glycans, as well as poly-N-acetyllactosamine chains. Adipogenesis was accompanied by an increased amount of biantennary fucosylated structures, decreased amount of fucosylated, afucosylated tri- and tetraantennary structures and increased sialylation. N-glycans H6N5F1 and H7N6F1 were significantly overexpressed in undifferentiated MSCs while H3N4F1 and H5N4F3 were upregulated in adipogenically differentiated MSCs. These glycan structures are promising candidate markers to detect and distinguish MSCs and their adipogenic progeny.
PMCID: PMC3856714  PMID: 23829188
3.  Mesenchymal stem cells and their chondrogenic differentiated and dedifferentiated progeny express chemokine receptor CCR9 and chemotactically migrate toward CCL25 or serum 
Guided migration of chondrogenically differentiated cells has not been well studied, even though it may be critical for growth, repair, and regenerative processes. The chemokine CCL25 is believed to play a critical role in the directional migration of leukocytes and stem cells. To investigate the motility effect of serum- or CCL25-mediated chemotaxis on chondrogenically differentiated cells, mesenchymal stem cells (MSCs) were induced to chondrogenic lineage cells.
MSC-derived chondrogenically differentiated cells were characterized for morphology, histology, immunohistochemistry, quantitative polymerase chain reaction (qPCR), surface profile, and serum- or CCL25-mediated cell migration. Additionally, the chemokine receptor, CCR9, was examined in different states of MSCs.
The chondrogenic differentiated state of MSCs was positive for collagen type II and Alcian blue staining, and showed significantly upregulated expression of COL2A1and SOX9, and downregulated expression of CD44, CD73, CD90, CD105 and CD166, in contrast to the undifferentiated and dedifferentiated states of MSCs. For the chondrogenic differentiated, undifferentiated, and dedifferentiated states of MSCs, the serum-mediated chemotaxis was in a percentage ratio of 33%:84%:85%, and CCL25-mediated chemotaxis was in percentage ratio of 12%:14%:13%, respectively. On the protein level, CCR9, receptor of CCL25, was expressed in the form of extracellular and intracellular domains. On the gene level, qPCR confirmed the expression of CCR9 in different states of MSCs.
CCL25 is an effective cue to guide migration in a directional way. In CCL25-mediated chemotaxis, the cell-migration rate was almost the same for different states of MSCs. In serum-mediated chemotaxis, the cell-migration rate of chondrogenically differentiated cells was significantly lower than that in undifferentiated or dedifferentiated cells. Current knowledge of the surface CD profile and cell migration could be beneficial for regenerative cellular therapies.
PMCID: PMC3854782  PMID: 23958031
Chondrogenically differentiated cells; Stem cells; Cell migration; Chemotaxis; CCL25; CCR9
4.  Reverse Differentiation as a Gene Filtering Tool in Genome Expression Profiling of Adipogenesis for Fat Marker Gene Selection and Their Analysis 
PLoS ONE  2013;8(7):e69754.
During mesenchymal stem cell (MSC) conversion into adipocytes, the adipogenic cocktail consisting of insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine not only induces adipogenic-specific but also genes for non-adipogenic processes. Therefore, not all significantly expressed genes represent adipogenic-specific marker genes. So, our aim was to filter only adipogenic-specific out of all expressed genes. We hypothesize that exclusively adipogenic-specific genes change their expression during adipogenesis, and reverse during dedifferentiation. Thus, MSC were adipogenic differentiated and dedifferentiated.
Adipogenesis and reverse adipogenesis was verified by Oil Red O staining and expression of PPARG and FABP4. Based on GeneChips, 991 genes were differentially expressed during adipogenesis and grouped in 4 clusters. According to bioinformatic analysis the relevance of genes with adipogenic-linked biological annotations, expression sites, molecular functions, signaling pathways and transcription factor binding sites was high in cluster 1, including all prominent adipogenic genes like ADIPOQ, C/EBPA, LPL, PPARG and FABP4, moderate in clusters 2–3, and negligible in cluster 4. During reversed adipogenesis, only 782 expressed genes (clusters 1–3) were reverted, including 597 genes not reported for adipogenesis before. We identified APCDD1, CHI3L1, RARRES1 and SEMA3G as potential adipogenic-specific genes.
The model system of adipogenesis linked to reverse adipogenesis allowed the filtration of 782 adipogenic-specific genes out of total 991 significantly expressed genes. Database analysis of adipogenic-specific biological annotations, transcription factors and signaling pathways further validated and valued our concept, because most of the filtered 782 genes showed affiliation to adipogenesis. Based on this approach, the selected and filtered genes would be potentially important for characterization of adipogenesis and monitoring of clinical translation for soft-tissue regeneration. Moreover, we report 4 new marker genes.
PMCID: PMC3724870  PMID: 23922792
5.  A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures 
BioResearch Open Access  2012;1(6):297-305.
Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×105 cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.
PMCID: PMC3559221  PMID: 23514965
cell isolation; chondrogenic differentiation; enzymatic pellet digestion; extracellular matrix
6.  A method to screen and evaluate tissue adhesives for joint repair applications 
Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications.
Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives.
Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa).
The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings.
PMCID: PMC3732078  PMID: 22984926
Tissue adhesive; Bonding strength; Cartilage; Bone; Transplant; Tissue engineering; Joint repair; Test method
7.  Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis 
PLoS ONE  2011;6(12):e28513.
Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis.
Methodology/Principal Findings
To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression.
We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.
PMCID: PMC3235117  PMID: 22174827
8.  Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development 
BMC Genomics  2011;12:461.
Adipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17), which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data.
The mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression) that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p < 0.00001). Subsequently, groups of up- or down-regulated genes were formed and analyzed with biochemical and cluster tools. Among the 184 genes, we identified already known transcription factors such as PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein.
Comparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for adipogenic differentiation. Our results encourage further and more focused studies on the functional relevance of particular adipogenic candidate genes.
PMCID: PMC3222637  PMID: 21943323
9.  Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation 
Arthritis Research & Therapy  2009;11(5):R133.
Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA.
Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score > 3, Ahlbäck Score > 2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using real-time PCR.
Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (ACAN, COL2A1, COMP, CRTL1, SOX9) and genes involved in matrix synthesis (BGN, CILP2, COL9A2, COL11A1, TIMP4) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (ALPL, COL1A1, COL3A1, COL10A1, MMP13, POSTN, PTH1R, RUNX2) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, was differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated.
Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA, and OA chondrocytes fulfill the requirements for matrix-associated ACT.
PMCID: PMC2787268  PMID: 19723327
10.  Tissue engineering in the rheumatic diseases 
Diseases such as degenerative or rheumatoid arthritis are accompanied by joint destruction. Clinically applied tissue engineering technologies like autologous chondrocyte implantation, matrix-assisted chondrocyte implantation, or in situ recruitment of bone marrow mesenchymal stem cells target the treatment of traumatic defects or of early osteoarthritis. Inflammatory conditions in the joint hamper the application of tissue engineering during chronic joint diseases. Here, most likely, cartilage formation is impaired and engineered neocartilage will be degraded. Based on the observations that mesenchymal stem cells (a) develop into joint tissues and (b) in vitro and in vivo show immunosuppressive and anti-inflammatory qualities indicating a transplant-protecting activity, these cells are prominent candidates for future tissue engineering approaches for the treatment of rheumatic diseases. Tissue engineering also provides highly organized three-dimensional in vitro culture models of human cells and their extracellular matrix for arthritis research.
PMCID: PMC2688224  PMID: 19232063
11.  Antirheumatic drug response signatures in human chondrocytes: potential molecular targets to stimulate cartilage regeneration 
Rheumatoid arthritis (RA) leads to progressive destruction of articular cartilage. This study aimed to disclose major mechanisms of antirheumatic drug action on human chondrocytes and to reveal marker and pharmacological target genes that are involved in cartilage dysfunction and regeneration.
An interactive in vitro cultivation system composed of human chondrocyte alginate cultures and conditioned supernatant of SV40 T-antigen immortalised human synovial fibroblasts was used. Chondrocyte alginate cultures were stimulated with supernatant of RA synovial fibroblasts, of healthy donor synovial fibroblasts, and of RA synovial fibroblasts that have been antirheumatically treated with disease-modifying antirheumatic drugs (DMARDs) (azathioprine, gold sodium thiomalate, chloroquine phosphate, and methotrexate), nonsteroidal anti-inflammatory drugs (NSAIDs) (piroxicam and diclofenac), or steroidal anti-inflammatory drugs (SAIDs) (methylprednisolone and prednisolone). Chondrocyte gene expression profile was analysed using microarrays. Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were performed for validation of microarray data.
Genome-wide expression analysis revealed 110 RA-related genes in human chondrocytes: expression of catabolic mediators (inflammation, cytokines/chemokines, and matrix degradation) was induced, and expression of anabolic mediators (matrix synthesis and proliferation/differentiation) was repressed. Potential marker genes to define and influence cartilage/chondrocyte integrity and regeneration were determined and include already established genes (COX-2, CXCR-4, IL-1RN, IL-6/8, MMP-10/12, and TLR-2) and novel genes (ADORA2A, BCL2-A1, CTGF, CXCR-7, CYR-61, HSD11B-1, IL-23A, MARCKS, MXRA-5, NDUFA4L2, NR4A3, SMS, STS, TNFAIP-2, and TXNIP). Antirheumatic treatment with SAIDs showed complete and strong reversion of RA-related gene expression in human chondrocytes, whereas treatment with NSAIDs and the DMARD chloroquine phosphate had only moderate to minor effects. Treatment with the DMARDs azathioprine, gold sodium thiomalate, and methotrexate efficiently reverted chondrocyte RA-related gene expression toward the 'healthy' level. Pathways of cytokine-cytokine receptor interaction, transforming growth factor-beta/Toll-like receptor/Jak-STAT (signal transducer and activator of transcription) signalling and extracellular matrix receptor interaction were targeted by antirheumatics.
Our findings indicate that RA-relevant stimuli result in the molecular activation of catabolic and inflammatory processes in human chondrocytes that are reverted by antirheumatic treatment. Candidate genes that evolved in this study for new therapeutic approaches include suppression of specific immune responses (COX-2, IL-23A, and IL-6) and activation of cartilage regeneration (CTGF and CYR-61).
PMCID: PMC2688247  PMID: 19192274
12.  Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study 
Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.
Human chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts (SF) derived from a normal donor (NDSF) and from a patient with RA (RASF), respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis (RMA) were used to identify differentially expressed genes. Expression of selected genes was verified by real-time RT-PCR.
Antibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators (IL-6, CCL2, CXCL1–3, CXCL8) released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (adenosine A2A receptor, cyclooxygenase-2), the NF-κB signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1–3, CXCL8, CCL20, CXCR4, IL-1β, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2).
Differential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic–anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of anti-rheumatic drugs on human chondrocyte gene expression.
PMCID: PMC2374452  PMID: 18205922
13.  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.
PMCID: PMC1906819  PMID: 17451597
14.  Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum*  
The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.
PMCID: PMC1599800  PMID: 16972324
Tissue engineering; Poly-lactic-co-glycolic acid polymer; Periosteum-derived progenitor cells; 3-dimensional culture
15.  Emerging strategies of bone and joint repair 
Arthritis Research  2000;2(6):433-436.
The advances in biomedicine over the past decade have provided revolutionary insights into molecules that mediate cell proliferation and differentiation. Findings on the complex interplay of cells, growth factors, matrix molecules and cell adhesion molecules in the process of tissue patterning have vitalized the revolutionary approach of bioregenerative medicine and tissue engineering. Here we review the impact of recent work in this interdisciplinary field on the treatment of musculoskeletal disorders. This novel concept combines the transplantation of pluripotent stem cells, and the use of specifically tailored biomaterials, arrays of bioactive molecules and gene transfer technologies to direct the regeneration of pathologically altered musculoskeletal tissues.
PMCID: PMC128870  PMID: 11094454
biomaterials; genetic engineering; morphogenic factors; tissue engineering

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