Meniscal Tissue Engineering Using Aligned Collagen Fibrous Scaffolds: Comparison of Different Human Cell Sources.

Hydrogel and electrospun scaffold materials support cell attachment and neotissue development and can be tuned to structurally and mechanically resemble native extracellular matrix by altering either electrospun fiber or hydrogel properties. In this study, we examined meniscus tissue generation from different human cell sources including meniscus cells derived from vascular and avascular regions, human bone marrow-derived mesenchymal stem cells, synovial cells, and cells from the infrapatellar fat pad (IPFP). All cells were seeded onto aligned electrospun collagen type I scaffolds and were optionally encapsulated in a tricomponent hydrogel. Single or multilayered constructs were generated and cultivated in defined medium with selected growth factors for 2 weeks. Cell viability, cell morphology, and gene-expression profiles were monitored using confocal microscopy, scanning electron microscopy, and quantitative polymerase chain reaction (qPCR), respectively. Multilayered constructs were examined with histology, immunohistochemistry, qPCR, and for tensile mechanical properties. For all cell types, TGFß1 and TGFß3 treatment increased COL1A1, COMP, Tenascin C (TNC), and Scleraxis (SCX) gene expression and deposition of collagen type I protein. IPFP cells generated meniscus-like tissues with higher meniscogenic gene expression, mechanical properties, and better cell distribution compared to other cell types studied. We show proof of concept that electrospun collagen scaffolds support neotissue formation and IPFP cells have potential for use in cell-based meniscus regeneration strategies.

Repair of Avascular Meniscus Tears with Electrospun Collagen Scaffolds Seeded with Human Cells.

The self-healing capacity of an injured meniscus is limited to the vascularized regions and is especially challenging in the inner avascular regions. As such, we investigated the use of human meniscus cell-seeded electrospun (ES) collagen type I scaffolds to produce meniscal tissue and explored whether these cell-seeded scaffolds can be implanted to repair defects created in meniscal avascular tissue explants. Human meniscal cells (derived from vascular and avascular meniscal tissue) were seeded on ES scaffolds and cultured. Constructs were evaluated for cell viability, gene expression, and mechanical properties. To determine potential for repair of meniscal defects, human meniscus avascular cells were seeded and cultured on aligned ES collagen scaffolds for 4 weeks before implantation. Surgical defects resembling "longitudinal tears" were created in the avascular zone of bovine meniscus and implanted with cell-seeded collagen scaffolds and cultured for 3 weeks. Tissue regeneration and integration were evaluated by histology, immunohistochemistry, mechanical testing, and magentic resonance imaging. Ex vivo implantation with cell-seeded collagen scaffolds resulted in neotissue that was significantly better integrated with the native tissue than acellular collagen scaffolds or untreated defects. Human meniscal cell-seeded ES collagen scaffolds may therefore be useful in facilitating meniscal repair of avascular meniscus tears.

Meniscus tissue engineering using a novel combination of electrospun scaffolds and human meniscus cells embedded within an extracellular matrix hydrogel.

Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are, therefore, likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time polymerase chain reaction (PCR), respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, and COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies.

Influence of cartilage extracellular matrix molecules on cell phenotype and neocartilage formation.

Interaction between chondrocytes and the cartilage extracellular matrix (ECM) is essential for maintaining the cartilage's role as a low-friction and load-bearing tissue. In this study, we examined the influence of cartilage zone-specific ECM on human articular chondrocytes (HAC) in two-dimensional and three-dimensional (3D) environments. Two culture systems were used. SYSTEM 1: HAC were cultured on cell-culture plates that had been precoated with the following ECM molecules for 7 days: decorin, biglycan, tenascin C (superficial zone), collagen type II, hyaluronan (HA) (middle and deep zones), and osteopontin (deep zone). Uncoated standard culture plates were used as controls. Expanded cells were examined for phenotypic changes using real-time polymerase chain reaction. In addition, expanded cells were placed into high-density pellet cultures for 14 days. Neocartilage formation was assessed via gene expression and histology evaluations. SYSTEM 2: HAC that were cultured on untreated plates and encapsulated in a 3D alginate scaffold were mixed with one of the zone-specific ECM molecules. Cell viability, gene expression, and histology assessments were conducted on 14-day-old tissues. In HAC monolayer culture, exposure to decorin, HA, and osteopontin increased COL2A1 and aggrecan messenger RNA (mRNA) levels compared with controls. Biglycan up-regulated aggrecan without a significant impact on COL2A1 expression; Tenascin C reduced COL2A1 expression. Neocartilage formed after preculture on tenascin C and collagen type II expressed higher COL2A1 mRNA compared with control pellets. Preculture of HAC on HA decreased both COL2A1 and aggrecan expression levels compared with controls, which was consistent with histology. Reduced proteoglycan 4 (PRG4) mRNA levels were observed in HAC pellets that had been precultured with biglycan and collagen type II. Exposing HAC to HA directly in 3D-alginate culture most effectively induced neocartilage formation, showing increased COL2A1 and aggrecan, and reduced COL1A1 compared with controls. Decorin treatments increased HAC COL2A1 mRNA levels. These data indicate that an appropriate exposure to cartilage-specific ECM proteins could be used to enhance cartilage formation and to even induce the formation of zone-specific phenotypes to improve cartilage regeneration.

Osteoarthritis: detection, pathophysiology, and current/future treatment strategies.

Osteoarthritis (OA) is a disease of the joint, and age is the major risk factor for its development. Clinical manifestation of OA includes joint pain, stiffness, and loss of mobility. Currently, no pharmacological treatments are available to treat this specific joint disease; only symptom-modifying drugs are available. Improvement in imaging technology, identification of biomarkers, and increased understanding of the molecular basis of OA will aid in detecting the early stages of disease. Yet the development of interventional strategies remains elusive and will be critical for effective prevention of OA-associated joint destruction. The potential of cell-based therapies may be applicable in improving joint function in mild to more advanced cases of OA. Ongoing studies to understand the basis of this disease will eventually lead to prevention and treatment strategies and will also be a key in reducing the social and economic burden of this disease. Nurses are advised to provide an integrative approach of disease assessment and management in OA patients' care with a focus on education and implementation. Knowledge and understanding of OA and how this affects the individual patient form the basis for such an integrative approach to all-round patient care and disease management.

Effects of perfusion and dynamic loading on human neocartilage formation in alginate hydrogels.

Dynamic loading and perfusion culture environments alone are known to enhance cartilage extracellular matrix (ECM) production in dedifferentiated articular chondrocytes. In this study, we explored whether a combination of these factors would enhance these processes over a free-swelling (FS) condition using adult human articular chondrocytes embedded in 2% alginate. The alginate constructs were placed into a bioreactor for perfusion (P) only (100 µL/per minute) or perfusion and dynamic compressive loading (PL) culture (20% for 1 h, at 0.5 Hz), each day. Control FS alginate gels were maintained in six-well static culture. Gene expression analysis was conducted on days 7 and 14, while cell viability, immunostaining, and mechanical property testing were performed on day 14 only. Total glycosaminoglycan (GAG) content and GAG synthesis were assessed after 14 days. Col2a1 mRNA expression levels were significantly higher (at least threefold; p<0.05) in both bioreactor conditions compared with FS by days 7 and 14. For all gene studies, no significant differences were seen between P and PL treatments. Aggrecan mRNA levels were not significantly altered in any condition although both GAG/DNA and (35)S GAG incorporation studies indicated higher GAG retention and synthesis in the FS treatment. Collagen type II protein deposition was low in all samples, link protein distribution was more diffuse in FS condition, and aggrecan deposition was located in the outer regions of the alginate constructs in both bioreactor conditions, yet more uniformly in the FS condition. Catabolic gene expression (matrix metalloproteinase 3 [MMP3] and inducible nitric oxide synthase [iNOS]) was higher in bioreactor conditions compared with FS, although iNOS expression levels decreased to approximately fourfold less than the FS condition by day 14. Our data indicate that conditions created in the bioreactor enhanced both anabolic and catabolic responses, similar to other loading studies. Perfusion was sufficient alone to promote this dual response. PL increased the deposition of aggrecan surrounding cells compared with the other conditions; however, overall low GAG retention in the bioreactor system was likely due to both perfusion and catabolic conditions created. Optimal conditions, which permit appropriate anabolic and catabolic processes for accumulation of ECM and tissue remodeling for neocartilage development, specifically for humans, are needed.