A Biodegradable Polymeric Matrix for the Repair of Annulus Fibrosus Defects in Intervertebral Discs.

BACKGROUND: Tissue defects in the annulus fibrosus (AF) due to intervertebral disc (IVD) degeneration or after nucleodiscectomy have little self-healing capacity. To prevent progressive degeneration of the IVD, the AF must be repaired. Biological closure has not yet been achieved and is a challenge for the research community. In this study, a scaffold made of absorbable poly (glycolic acid) (PGA) and hyaluronan (HA) that exhibit excellent biocompatibility and cell colonization properties was used to repair AF defects in an ovine model. METHODS: A partial resection was performed in AF in L3/4 or L4/5 of 10 sheep and PGA-HA scaffolds were implanted on the defects (n = 5), while defects in the control group were left untreated (n = 5). Three months post-operation, the lumbar discs were sectioned and stained with hematoxylin and eosin and safranin-O/fast-green. Histological features including proteoglycan content, annular structure, cellular morphology, blood vessel ingrowth and tear/cleft formation were scored using a modified scoring scheme by 3 investigators and evaluated by a pathologist independently. RESULTS: The treated AF exhibited significantly enhanced repair tissue structure with signs of proteoglycan formation compared to the untreated group. The median scores were 4.3 for the treated and 9.8 for the untreated group. Cystic degeneration, perivascular infiltration, inflammation and necrosis were only present in the untreated group. Blood vessel ingrowth and tear/cleft formation were increased, though not significant, in the untreated group while cell morphology was comparable in both groups. CONCLUSION: PGA-HA scaffolds used for AF closure support repair tissue formation in an ovine lumbar disc defect model.

Blends of gelatin and hyaluronic acid stratified by stereolithographic bioprinting approximate cartilaginous matrix gradients.

Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage-like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell-laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient-like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades.

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor ß1 (TGFß1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFß1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

Meniscus-shaped cell-free polyglycolic acid scaffold for meniscal repair in a sheep model.

Since loss of meniscus is correlated with an increasing risk for osteoarthritis, meniscal scaffolds are proposed as new strategies. Development of a suitable scaffold has to take into account differing meniscus thickness, exposure to compressive and tensile forces combined with high porosity and biocompatibility of the material. After physical testing of three flat scaffolds composed of different modified polyglycolic acid (PGA) fibers, a three-dimensional meniscus-shaped PGA-hyaluronan implant was generated. Micro-computed tomography showed 90% porosity in the outer area with 50% in the inner area of the implant. Biocompatibility and expression of meniscus typical cartilaginous genes were shown for human meniscus cells cultivated in the implant with 10% human serum or 5% platelet-rich plasma for 14 days in vitro. The proof-of-concept study in sheep demonstrated proteoglycan- and collagen type I-rich repair tissue formation in partial meniscectomy combined with a meniscus-shaped PGA-hyaluronan implant after 6 months. In contrast, the control showed nearly no repair tissue formation. Thus, meniscus-shaped PGA-hyaluronan implants might be a suitable therapeutic approach to support repair tissue formation in partial meniscectomy.

Proliferation, Migration, and ECM Formation Potential of Human Annulus Fibrosus Cells Is Independent of Degeneration Status.

OBJECTIVE: The objective was to evaluate the proliferating, migratory and extracellular matrix (ECM) forming potential of annulus fibrosus cells derived from early (edAFC) or advanced (adAFC) degenerative tissue and their usability as a possible cell source for regenerative approaches for AF closure. DESIGN: EdAFC (n = 5 Pfirrman score of 2-3) and adAFC (n = 5 Pfirrman score of 4-5) were isolated from tissue of patients undergoing spine stabilizing surgery. Cell migration on stimulation with human serum (HS), platelet-rich plasma (PRP), and transforming growth factor ß-3 (TGFB3) was assessed by migration assay and proliferation was assessed on stimulation with HS. Induction of ECM synthesis was evaluated by gene expression analysis of AF-related genes in three-dimensional scaffold cultures that have been stimulated with 5% PRP or 10 ng/mL TGFB3 and histologically by collagen type I, type II, alcian blue, and safranin-O staining. RESULTS: EdAFC and adAFC were significantly attracted by 10% HS and 5% PRP. Additionally, both cell groups proliferated under stimulation with HS. Stimulation with 10 ng/mL TGFB3 showed significant induction of gene expression of collagen type II and aggrecan, while 5% PRP decreased the expression of collagen type I. Both cell groups showed formation of AF-like ECM after stimulation with TGFB3, whereas stimulation with PRP did not. CONCLUSIONS: Our study demonstrated that AF cells retain their potential for proliferation, migration, and ECM formation independent of the degeneration status of the tissue. Proliferation, migration, and ECM synthesis of the endogenous AF cells can be supported by different supplements. Hence, endogenous AF cells might be a suitable cell source for a regenerative repair approaches.

Photopolymerizable gelatin and hyaluronic acid for stereolithographic 3D bioprinting of tissue-engineered cartilage.

To create artificial cartilage in vitro, mimicking the function of native extracellular matrix (ECM) and morphological cartilage-like shape is essential. The interplay of cell patterning and matrix concentration has high impact on the phenotype and viability of the printed cells. To advance the capabilities of cartilage bioprinting, we investigated different ECMs to create an in vitro chondrocyte niche. Therefore, we used methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) in a stereolithographic bioprinting approach. Both materials have been shown to support cartilage ECM formation and recovery of chondrocyte phenotype. We used these materials as bioinks to create cartilage models with varying chondrocyte densities. The models maintained shape, viability, and homogenous cell distribution over 14 days in culture. Chondrogenic differentiation was demonstrated by cartilage-typical proteoglycan and type II collagen deposition and gene expression (COL2A1, ACAN) after 14 days of culture. The differentiation pattern was influenced by cell density. A high cell density print (25 × 106 cells/mL) led to enhanced cartilage-typical zonal segmentation compared to cultures with lower cell density (5 × 106 cells/mL). Compared to HAMA, GelMA resulted in a higher expression of COL1A1, typical for a more premature chondrocyte phenotype. Both bioinks are feasible for printing in vitro cartilage with varying differentiation patterns and ECM organization depending on starting cell density and chosen bioink. The presented technique could find application in the creation of cartilage models and in the treatment of articular cartilage defects using autologous material and adjusting the bioprinted constructs size and shape to the patient. © 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2649-2657, 2019.

Characterization of plasma fibronectin for migration, proliferation, and differentiation on human articular chondrocytes.

Plasma fibronectin (pFN) plays a crucial role in wound healing by binding to integrins and inducing cell migration. It is known to induce the migration and proliferation of mesenchymal progenitor cells in vitro, which play a key role during microfracture in cartilage repair. Endogenous chondrocytes from the native cartilage of the defect rim might aid in cartilage repair. In this study, the effect of pFN on proliferation, migration, and differentiation was tested on human articular chondrocytes. Results showed that treatment with pFN increased the migration of chondrocytes in a range of 1-30 µg/ml as tested with no effect on proliferation. TGFß3-induced chondrogenesis was not affected by pFN. Especially, gene expression of matrix metalloproteinases was not increased by pFN. Plasma FN fragmentation due to storage conditions could be excluded by SDS-PAGE. Moreover, bioactivity of pFN did not alter during storage at 4°C and 40°C for up to 14 days. Taken together, pFN induces the migration but not proliferation of human articular chondrocytes with no inhibitory effect on chondrogenic differentiation. Additionally, no loss of activity or fragmentation of pFN was observed after lyophilization and storage, making pFN an interesting bioactive factor for chondrocyte recruitment.

Chemokine CCL25 Induces Migration and Extracellular Matrix Production of Anulus Fibrosus-Derived Cells.

Intervertebral disc degeneration is a major source of back pain. For intervertebral disc regeneration after herniation a fast closure of anulus fibrosus (AF) defects is crucial. Here, the use of the C-C motif chemokine ligand 25 (CCL)25 in comparison to differentiation factors such as transforming growth factor (TGF)ß3, bone morphogenetic protein (BMP)2, BMP7, BMP12, and BMP14 (all in concentrations of 10, 50 and 100 ng/mL) was tested in an in vitro micro mass pellet model with isolated and cultivated human AF-cells (n = 3) to induce and enhance AF-matrix formation. The pellets were differentiated (serum-free) with supplementation of the factors. After 28 days all used factors induced proteoglycan production (safranin O staining) and collagen type I production (immunohistochemical staining) in at least one of the tested concentrations. Histomorphometric scoring revealed that TGFß3 delivered the strongest induction of proteoglycan production in all three concentrations. Furthermore, it was the only factor able to facilitate collagen type II production, even higher than in native tissue samples. CCL25 was also able to induce proteoglycan and collagen type I production comparable to several BMPs. CCL25 could additionally induce migration of AF-cells in a chemotaxis assay and therefore possibly aid in regeneration processes after disc herniation by recruiting AF-cells.

Proliferation, migration and differentiation potential of human mesenchymal progenitor cells derived from osteoarthritic subchondral cancellous bone.

Background: For regenerative therapies in the orthopedic field, one prerequisite for therapeutic success in the treatment of cartilage defects is the potential of body's own cells to migrate, proliferate and differentiate into functional cells. While this has been demonstrated for mesenchymal stem and progenitor cells (MPC) from healthy tissue sources, the potential of cells from degenerative conditions is unclear. In this study the regenerative potential of MPC derived from subchondral cancellous bone with diagnosed osteoarthritis is evaluated in vitro. Methods: OaMPC isolated from bone chips of three individual patients with Kellgren grade 3 osteoarthritis were characterized by analysis of cell surface antigen pattern. Cell proliferation was evaluated by doubling time and population doubling rate. Cell migration was assessed using a multi-well migration assay. Multi-lineage potential was evaluated by histological staining of adipogenic, osteogenic and chondrogenic markers. In addition, chondrogenic differentiation was verified by qPCR. Results: OaMPC showed a stable proliferation and a typical surface antigen pattern known from mesenchymal stem cells. Cell migration of oaMPC can be induced by human blood serum. OaMPC were capable of adipogenic, osteogenic and chondrogenic differentiation comparable to MPC derived from healthy conditions. Conclusion: OaMPC derived from knee joints affected by osteoarthritic conditions showed regeneration potential regarding migration, proliferation and chondrogenic differentiation. This suggests that oaMPC are able to contribute to cartilage repair tissue formation.

Effect of serum and platelet-rich plasma on human early or advanced degenerative meniscus cells.

PURPOSE: The purpose of this in vitro study was to evaluate the migratory, proliferating, and extracellular matrix (ECM) forming effect of human serum (HS) and platelet-rich plasma (PRP) on meniscus cells derived from human knees with early or advanced degenerative changes. MATERIALS AND METHODS: Medial menisci from knees with early degenerative changes (n = 5; mean Kellgren score of 1) undergoing arthroscopic meniscal surgery and advanced degenerative changes (n = 5; mean Kellgren score of 4) undergoing total knee replacement were collected. Cell migration and proliferation upon stimulation with HS and PRP were assessed by migration and proliferation assays. Induction of meniscal ECM was evaluated histologically by hematoxylin and eosin, collagen type I, and alcian blue staining and by gene expression analysis of meniscus-related genes in pellets that have been stimulated with 10% HS or 5% PRP. RESULTS: Meniscal cells from knees with early and advanced degenerative changes were significantly attracted by 2.5%-30% PRP or 10% HS. Cell proliferation was significantly increased upon stimulation with 10% HS or 5% PRP. Both cell groups showed the formation of a well-structured, meniscus-like ECM after stimulation with 10% HS, whereas stimulation with 5% PRP led to inhomogeneous, more fibrous ECM. Stimulation with 10% HS showed a significant induction of aggrecan and COMP, while 5% PRP showed no inducing effect. CONCLUSIONS: Only stimulation with HS showed the formation of meniscal ECM as well as cell proliferating and migratory effects on meniscal cells derived from knees with early or advanced degenerative changes. Thus, we suggest that the selected stimulating factor itself and not the status of the knee may primarily affect repair processes. HS may have a potential to augment in meniscal repair procedures.

Effect of Human Serum and 2 Different Types of Platelet Concentrates on Human Meniscus Cell Migration, Proliferation, and Matrix Formation.

PURPOSE: To evaluate the effect of 10% human serum (HS), 5% platelet-rich plasma (PRP), and 5% autologous conditioned plasma (ACP) on migration, proliferation, and extracellular matrix (ECM) synthesis of human meniscus cells. METHODS: Cell migration and proliferation on stimulation with HS, PRP, and ACP were assessed by chemotaxis assays and measurement of genomic DNA content. Meniscus cells were cultivated in pellets stimulated with 10% HS, 5% PRP, or 5% ACP. Meniscal ECM formation was evaluated by histochemical staining of collagen type I, type II, and proteoglycans and by analysis of fibrochondrocyte marker gene expression. RESULTS: Human meniscus cells were significantly attracted by all 3 blood-derived products (10% HS and 5% ACP: P = .0001, 5% PRP: P = .0002). Cell proliferation at day 9 was significantly increased on stimulation with 10% HS (P = .0001) and 5% PRP (P = .0002) compared with 5% ACP and controls. Meniscus cell pellet cultures showed the formation of a well-structured meniscal ECM with deposition of collagen type I, type II, and proteoglycans on stimulation with 10% HS, whereas 5% PRP or 5% ACP resulted in the formation of an inhomogeneous and more fibrous ECM. Stimulation with 10% HS and 5% ACP showed a significant induction of fibrochondrocyte marker genes such as aggrecan (HS: P = .0002, ACP: P = .0147), cartilage oligomeric matrix protein (HS: P = .0002, ACP: P = .0005), and biglycan (HS: P = .0002, ACP: P = .0003), whereas PRP showed no inducing effect. CONCLUSIONS: Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches. CLINICAL RELEVANCE: Our findings may suggest that HS might be a beneficial augment for meniscus repair.

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

PURPOSE: To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. METHODS: Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. RESULTS: MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. CONCLUSIONS: Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. CLINICAL RELEVANCE: Our findings may help to explain the variability of results in studies examining the use of PRP clinically.

Fibronectin stimulates migration and proliferation, but not chondrogenic differentiation of human subchondral progenitor cells.

AIMS: To evaluate the impact of human plasma-derived fibronectin (FN) on human subchondral mesenchymal progenitor cells regarding cell migration, proliferation, and chondrogenic differentiation. MATERIALS & METHODS: Human subchondral mesenchymal progenitor cells were analyzed for their migration capacity upon treatment with human plasma-derived FN. Proliferation activity was evaluated by DNA content. For chondrogenesis, cells were cultured in high-density pellet cultures in the presence of FN, TGFß3, and a combination thereof. RESULTS: Treatment of progenitors with FN significantly increased the number of migrating cells and elevated proliferative activity. Histological staining indicated formation of an extracellular matrix with type II collagen. Gene expression analysis gave no evidence for chondrogenic differentiation mediated by FN, but revealed a significant induction of type II collagen expression. CONCLUSION: FN has a potential to recruit human subchondral mesenchymal progenitor cells, possibly supporting proliferation and matrix assembly in cartilage repair procedures using bioactive implants after microfracture treatment.

Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells.

BACKGROUND: Three-dimensional (3D) culture in porous biomaterials as well as stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated, if an initial boost with transforming growth factor-beta 1 (TGF-beta 1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells. METHODS: Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of non-absorbable polyvinylidene fluoride. An initial boost of TGF-beta 1 or TGF-beta 1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan and type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13. RESULTS: An initial boost with TGF-beta 1 or TGF-beta 1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from both, slightly degenerated, or severely degenerated tissue are capable of significant up-regulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower up-regulations in some matrix molecules such as aggrecan. CONCLUSIONS: We failed to show a supportive effect of an initial boost with TGF-beta 1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems.

Initial boost release of transforming growth factor-ß3 and chondrogenesis by freeze-dried bioactive polymer scaffolds.

In cartilage regeneration, bio-activated implants are used in stem and progenitor cell-based microfracture cartilage repair procedures. Our aim was to analyze the chondrogenic potential of freeze-dried resorbable polymer-based polyglycolic acid (PGA) scaffolds bio-activated with transforming growth factor-ß3 (TGFB3) on human subchondral mesenchymal progenitor cells known from microfracture. Progenitor cells derived from femur heads were cultured in the presence of freeze-dried TGFB3 in high-density pellet culture and in freeze-dried TGFB3-PGA scaffolds for chondrogenic differentiation. Progenitor cell cultures in PGA scaffolds as well as pellet cultures with and without continuous application of TGFB3 served as controls. Release studies showed that freeze-dried TGFB3-PGA scaffolds facilitate a rapid, initial boost-like release of 71.5% of TGFB3 in the first 10 h. Gene expression analysis and histology showed induction of typical chondrogenic markers like type II collagen and formation of cartilaginous tissue in TGFB3-PGA scaffolds seeded with subchondral progenitor cells and in pellet cultures stimulated with freeze-dried TGFB3. Chondrogenic differentiation in freeze-dried TGFB3-PGA scaffolds was comparable to cultures receiving TGFB3 continuously, while non-stimulated controls did not show chondrogenesis during prolonged culture for 14 days. These results suggest that bio-activated, freeze-dried TGFB3-PGA scaffolds have chondrogenic potential and are a promising tool for stem cell-mediated cartilage regeneration.

Suitability of porcine chondrocyte micromass culture to model osteoarthritis in vitro.

In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.

Human platelet-rich plasma induces chondrogenic differentiation of subchondral progenitor cells in polyglycolic acid-hyaluronan scaffolds.

Cartilage repair approaches may be improved by addition of human platelet-rich plasma (PRP) that increases chondrogenic differentiation of mesenchymal stem and progenitor cells. The aim of our study was to evaluate the effect of human PRP on the differentiation of multipotent human subchondral progenitor cells in resorbable polyglycolic acid-hyaluronan (PGA-HA) scaffolds. PGA-HA scaffolds were loaded with subchondral progenitor cells and stimulated with transforming growth factor-beta3 (TGFB3) or 5% PRP, whereas nonstimulated cultures served as controls. Chondrogenic differentiation was evaluated by real-time gene expression analysis of typical chondrogenic marker genes and by immunohistochemical staining of extracellular cartilage matrix molecules such as proteoglycans and collagen type II. TGFB3 and PRP induced the expression of chondrogenic marker genes collagen type II and IX, aggrecan, and cartilage oligomeric matrix protein in subchondral progenitor cells cultured in PGA-HA scaffolds compared with nonstimulated controls. Progenitor cells in PGA-HA scaffolds formed an extracellular matrix rich in proteoglycans and collagen type II on treatment with PRP, but to a lesser extent, than in cultures stimulated with TGFB3. The results suggest that PRP induces chondrogenic differentiation of progenitor cells in PGA-HA scaffolds and may be therefore beneficial in scaffold-assisted cartilage repair approaches involving stem and progenitor cells.

Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants.

The small size and heterogeneity of the pores in bacterial nanocellulose (BNC) hydrogels limit the ingrowth of cells and their use as tissue-engineered implant materials. The use of placeholders during BNC biosynthesis or post-processing steps such as (touch-free) laser perforation can overcome this limitation. Since three-dimensionally arranged channels may be required for homogeneous and functional seeding, three-dimensional (3-D) laser perforation of never-dried BNC hydrogels was performed. Never-dried BNC hydrogels were produced in different shapes by: (i) the cultivation of Gluconacetobacter xylinus (DSM 14666; synonym Komagataeibacter xylinus) in nutrient medium; (ii) the removal of bacterial residues/media components (0.1M NaOH; 30 min; 100 °C) and repeated washing (deionized water; pH 5.8); (iii) the unidirectional or 3-D laser perforation and cutting (pulsed CO2 Rofin SC × 10 laser; 220 µm channel diameter); and (iv) the final autoclaving (2M NaOH; 121 °C; 20 min) and washing (pyrogen-free water). In comparison to unmodified BNC, unidirectionally perforated--and particularly 3-D-perforated - BNC allowed ingrowth into and movement of vital bovine/human chondrocytes throughout the BNC nanofiber network. Laser perforation caused limited structural modifications (i.e. fiber or globular aggregates), but no chemical modifications, as indicated by Fourier transform infrared spectroscopy, X-ray photoelectron scattering and viability tests. Pre-cultured human chondrocytes seeding the surface/channels of laser-perforated BNC expressed cartilage-specific matrix products, indicating chondrocyte differentiation. 3-D-perforated BNC showed compressive strength comparable to that of unmodified samples. Unidirectionally or 3-D-perforated BNC shows high biocompatibility and provides short diffusion distances for nutrients and extracellular matrix components. Also, the resulting channels support migration into the BNC, matrix production and phenotypic stabilization of chondrocytes. It may thus be suitable for in vivo application, e.g. as a cartilage replacement material.

Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell-free resorbable polymer scaffolds.

Degeneration of intervertebral discs (IVDs) occurs frequently and is often associated with lower back pain. Recent treatment options are limited and treat the symptoms rather than regenerate the degenerated disc. Cell-free, freeze-dried resorbable polyglycolic acid (PGA)-hyaluronan implants were used in an ovine IVD degeneration model. The nucleus pulposus of the IVD was partially removed, endoscopically. PGA-hyaluronan implants were immersed in autologous sheep serum and implanted into the disc defect. Animals with nucleotomy only served as controls. The T2-weighted/fat suppression sequence signal intensity index of the operated discs, as assessed by magnetic resonance imaging (MRI), showed that implantation of the PGA-hyaluronan implant improved (p = 0.0066) the MRI signal compared to controls at 6 months after surgery. Histological analysis by haematoxylin and eosin and safranin O staining showed the ingrowth of cells with typical chondrocytic morphology, even cell distribution, and extracellular matrix rich in proteoglycan. Histomorphometric analyses confirmed that the implantation of the PGA-hyaluronan scaffolds improved (p = 0.027) the formation of regenerated tissue after nucleotomy. Disc heights remained stable in discs with nucleotomy only as well as after implantation of the implant. In conclusion, implantation of cell-free polymer-based implants after nucleotomy induces nucleus pulposus tissue regeneration and improves disc water content in the ovine model.

Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenic differentiation in vitro and cartilage repair in the rabbit model.

In cartilage repair, scaffold-assisted one-step approaches are used to improve the microfracture (Mfx) technique. Since the number of progenitors in Mfx is low and may further decrease with age, aim of our study was to analyze the chondrogenic potential of freeze-dried polyglycolic acid-hyaluronan (PGA-HA) implants preloaded with mesenchymal stem cells (MSCs) in vitro and in a rabbit articular cartilage defect model. Human bone marrow-derived MSC from iliac crest were cultured in freeze-dried PGA-HA implants for chondrogenic differentiation. In a pilot study, implants were loaded with autologous rabbit MSC and used to cover 5 mm × 6 mm full-thickness femoral articular cartilage defects (n = 4). Untreated defects (n = 3) served as controls. Gene expression analysis and histology showed induction of typical chondrogenic marker genes like type II collagen and formation of hyaline-like cartilaginous tissue in MSC-laden PGA-HA implants. Histological evaluation of rabbit repair tissue formation after 30 and 45 days showed formation of repair tissue, rich in chondrocytic cells and of a hyaline-like appearance. Controls showed no articular resurfacing, tissue repair in the subchondral zone and fibrin formation. These results suggest that MSC-laden PGA-HA scaffolds have chondrogenic potential and are a promising option for stem cell-mediated cartilage regeneration.