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The purpose of this study was to evaluate the quality of surface contouring of chondromalacic cartilage by bipolar radio frequency energy using different treatment patterns in an animal model, as well as examining the impact of the treatment onto chondrocyte viability by two different methods. Our experiments were conducted on 36 fresh osteochondral sections from the tibia plateau of slaughtered 6-month-old pigs, where the thickness of the cartilage is similar to that of human wrist cartilage. An area of 1 cm 2 was first treated with emery paper to simulate the chondromalacic cartilage. Then, the treatment with RFE followed in 6 different patterns. The osteochondral sections were assessed for cellular viability (live/dead assay, caspase (cell apoptosis marker) staining, and quantitative analysed images obtained by fluorescent microscopy). For a quantitative characterization of none or treated cartilage surfaces, various roughness parameters were measured using confocal laser scanning microscopy (Olympus LEXT OLS 4000 3D). To describe the roughness, the Root-Mean-Square parameter (Sq) was calculated. A smoothing effect of the cartilage surface was detectable upon each pattern of RFE treatment. The Sq for native cartilage was Sq = 3.8 ± 1.1  μ m. The best smoothing pattern was seen for two RFE passes and a 2-second pulsed mode (B2p2) with an Sq = 27.3 ± 4.9  μ m. However, with increased smoothing, an augmentation in chondrocyte death up to 95% was detected. Using bipolar RFE treatment in arthroscopy for small joints like the wrist or MCP joints should be used with caution. In the case of chondroplasty, there is a high chance to destroy the joint cartilage., Competing Interests: All authors declare that there are no competing interests regarding the publication of this paper., (Copyright © 2020 Michaela Huber et al.)

The endogenous healing potential of avascular meniscal lesions is poor. Up to now, partial meniscectomy is still the treatment of choice for meniscal lesions within the avascular area. However, the large loss of meniscus substance predisposes the knee for osteoarthritic changes. Tissue engineering techniques for the replacement of such lesions could be a promising alternative treatment option. Thus, a polyurethane scaffold, which is already in clinical use, loaded with mesenchymal stromal cells, was analyzed for the repair of critical meniscus defects in the avascular zone. Large, approximately 7 mm broad meniscus lesions affecting both the avascular and vascular area of the lateral rabbit meniscus were treated with polyurethane scaffolds either loaded or unloaded with mesenchymal stromal cells. Menisci were harvested at 6 and 12 weeks after initial surgery. Both cell-free and cell-loaded approaches led to well-integrated and stable meniscus-like repair tissue. However, an accelerated healing was achieved by the application of mesenchymal stromal cells. Dense vascularization was detected throughout the repair tissue of both treatment groups. Overall, the polyurethane scaffold seems to promote the vessel ingrowth. The application of mesenchymal stromal cells has the potential to speed up the healing process.

Background: Treatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis., Methods: A rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis., Results: After resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p < 0.05)., Conclusions: The regenerative potential of the meniscus by an autologous cell-based tissue engineering approach was shown even in a challenging setting of early osteoarthritis. Autologous MSCs and meniscal cells were found to have improved meniscal healing in an animal model, thus demonstrating their feasibility in a clinical setting. However, donor site morbidity, reduced availability, and reduced chondrogenic differentiation of human meniscal cells from debris of meniscal tears favors autologous MSCs for clinical use for cell-based meniscus regeneration.

Mesenchymal stem cells (MSCs) seeded on specific carrier materials are a promising source for the repair of traumatic cartilage injuries. The best supportive carrier material has not yet been determined. As natural components of cartilage's extracellular matrix, hyaluronic acid and collagen are the focus of biomaterial research. In order to optimize chondrogenic support, we investigated three different scaffold compositions of a hyaluronic acid (HA)-gelatin based biomaterial., Methods: Human MSCs (hMSCs) were seeded under vacuum on composite scaffolds of three different HA-gelatin ratios and cultured in chondrogenic medium for 21 days. Cell-scaffold constructs were assessed at different time points for cell viability, gene expression patterns, production of cartilage-specific extracellular matrix (ECM) and for (immuno-)histological appearance. The intrinsic transforming growth factor beta (TGF-beta) uptake of empty scaffolds was evaluated by determination of the TGF-beta concentrations in the medium over time., Results: No significant differences were found for cell seeding densities and cell viability. hMSCs seeded on scaffolds with higher ratios of HA showed better cartilage-like differentiation in all evaluated parameters. TGF-beta uptake did not differ between empty scaffolds., Conclusion: Higher ratios of HA support the chondrogenic differentiation of hMSCs seeded on a HA-gelatin composite scaffold.

A combination of mesenchymal stem cells (MSCs) and scaffolds seems to be a promising approach for meniscus repair. To facilitate the search for an appropriate scaffold material a reliable and objective in vitro testing system is essential. This paper introduces a new scoring for this purpose and analyzes a hyaluronic acid (HA) gelatin composite scaffold and a polyurethane scaffold in combination with MSCs for tissue engineering of meniscus. The pore quality and interconnectivity of pores of a HA gelatin composite scaffold and a polyurethane scaffold were analyzed by surface photography and Berliner-Blau-BSA-solution vacuum filling. Further the two scaffold materials were vacuum-filled with human MSCs and analyzed by histology and immunohistochemistry after 21 days in chondrogenic media to determine cell distribution and cell survival as well as proteoglycan production, collagen type I and II content. The polyurethane scaffold showed better results than the hyaluronic acid gelatin composite scaffold, with signs of central necrosis in the HA gelatin composite scaffolds. The polyurethane scaffold showed good porosity, excellent pore interconnectivity, good cell distribution and cell survival, as well as an extensive content of proteoglycans and collagen type II. The polyurethane scaffold seems to be a promising biomaterial for a mesenchymal stem cell-based tissue engineering approach for meniscal repair. The new score could be applied as a new standard for in vitro scaffold testing.

Background: The application of radiofrequency energy (RFE) has become widespread for surgical performed chondroplasty especially due to the anticipated sealing effect, however the safety of this procedure in the wrist remains unclear. The purpose of this study was to investigate the subchondral temperature during radiofrequency energy (RFE) application simulating chondroplasty in an arthroscopic setting of the wrist., Methods: A chondroplasty of the lunate fossa was performed during an arthroscopy setting on 14 cadaver arms using monopolar or bipolar RFE. The temperature was recorded simultaneously from 7 predefined anatomical landmarks., Results: The mean temperature for both application modes did not exceed more than 30°C at all measured points, except for the lunate fossa. The highest subchondral measured peak temperature was 49.35°C (monopolar) and 69.21°C (bipolar) in the lunate fossa. In addition, the temperature decreased for both radiofrequency (RF) devices depending on the distance of the sensors to the RF-probe., Conclusion: It remains to be questionable how safe RFE can be used for chondroplasty in wrist arthroscopy under continuous irrigation and constant movement to obtain the desired sealing effect. However, the bipolar device should be applied with more caution since peak temperature in the lunate fossa almost reached 70°C even under continuous irrigation.

The transcription factor AP-2ε (activating enhancer-binding protein epsilon) is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4) strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1), the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2'-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

Meniscus integrity is the key for joint health of the knee. Therefore, the main goal of every meniscus treatment should be the maintenance of as much meniscus tissue as possible.Repair of meniscus tears can be achieved by meniscus suture. However, in a recently published meta-analysis, the long-term outcome of meniscus repair showed a mean failure rate of 24%.In a preclinical trial, locally applied mesenchymal stem cells produced differentiated meniscus-like tissue in meniscus tears indicating that mesenchymal-based cells, harvested from the bone marrow, enhance meniscus healing in critical-size meniscus tears.Symptomatic meniscus defects offer the option for meniscus transplantation with porous cell free biomaterials, when a complete meniscus rim is available. Cell-free biomaterials, which are actually in clinical application, reveal variable outcome in mid-term results from complete failure to regeneration with meniscus-like tissue.In several preclinical studies with different critical-size defects in the meniscus, the application of mesenchymal stem cells could significantly enhance meniscus regeneration compared to empty defects or to cell-free biomaterials.Regenerative treatment of meniscus with mesenchymal stem cells seems to be a promising approach to treat meniscal tears and defects. However it is still not clear, whether the stem cell effect is a direct action of the mesenchymal-based cells or is rather mediated by secretion of certain stimulating factors. The missing knowledge of the underlying mechanism is one of the reasons for regulatory burdens to permit these stem cell-based strategies in clinical practice. Other limitations are the necessity to expand cells prior to transplantation resulting in high treatment costs. Alternative treatment modalities, which use growth factors concentrated from peripheral blood aspirates or mononucleated cells concentrated from bone marrow aspirates, are currently in development in order to allow an attractive one-step procedure without the need for cell expansion in cultures and thus lower efforts and costs.In summary, Tissue Engineering of meniscus with mesenchymal based cells seems to be a promising approach to treat meniscal tears and defects in order to restore native meniscus tissue. However, advances of the technology are necessary to allow clinical application of this modern regenerative therapy.

Meniscal lesions in the avascular zone are still a problem in traumatology. Tissue Engineering approaches with mesenchymal stem cells (MSCs) showed successful regeneration of meniscal defects in the avascular zone. However, in daily clinical practice, a single stage regenerative treatment would be preferable for meniscus injuries. In particular, clinically applicable bioactive substances or isolated growth factors like platelet-rich plasma (PRP) or bone morphogenic protein 7 (BMP7) are in the focus of interest. In this study, the effects of PRP and BMP7 on the regeneration of avascular meniscal defects were evaluated. In vitro analysis showed that PRP secretes multiple growth factors over a period of 8 days. BMP7 enhances the collagen II deposition in an aggregate culture model of MSCs. However applied to meniscal defects PRP or BMP7 in combination with a hyaluronan collagen composite matrix failed to significantly improve meniscus healing in the avascular zone in a rabbit model after 3 months. Further information of the repair mechanism at the defect site is needed to develop special release systems or carriers for the appropriate application of growth factors to support biological augmentation of meniscus regeneration.

Background: Matrix-guided autologous chondrocyte transplantation (MACT) has been proposed as an option for treating large full-thickness cartilage defects. However, little is known about the chondrogenic potential of transplants for MACT at the time of implantation, although cell quality and chondrogenic differentiation of the implants are crucial for restoration of function after MACT., Questions/purposes: We therefore asked: (1) Do MACT implants allow deposition of extracellular cartilage matrix in an in vitro culture model? (2) Are these implants associated with improved knee function 1 year after MACT in large cartilage defects?, Methods: We retrospectively reviewed all 125 patients with large localized cartilage defects (mean defect size 5 cm(2)) of the knee who were treated with MACT from 2005 to 2010. The mean age was 31 years (range, 16-53 years). Portions of the cell-matrix constructs (n = 50) that were not implanted in the cartilage defects were further cultured and tested for their potential to form articular cartilage. Knee function of all patients was analyzed preoperatively, 3 months, and 1 year postoperatively with the International Knee Documentation Committee (IKDC) score., Results: In vitro assessment of the cell-matrix implants showed chondrogenic differentiation with positive staining for glycosaminoglycans and collagen II in all cultures. Enzyme-linked immunosorbent assay showed an increase of collagen II production. We observed an improvement in median IKDC score from 41 to 67 points at last followup., Conclusions: Cartilage extracellular matrix deposition shows adequate implant quality for MACT at the time of implantation and justifies the use for treatment of large cartilage defects.

Background: Intraarticular injections of local anesthetics are frequently used as part of multimodal pain regimens. However, recent data suggest that local anesthetics affect chondrocyte viability. In this study, we assessed the chondrotoxic effects of mepivacaine, ropivacaine, and bupivacaine. We hypothesized that specific cytotoxic potencies directly correlate with analgesic potencies, and that cytotoxic effects in intact cartilage are different than in osteoarthritic tissue., Methods: Human articular chondrocytes were exposed to equal and equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 hour. Cell viability, apoptosis, and necrosis were determined at predefined time points using flow cytometry, live-dead staining, and caspase detection. Intact and osteoarthritic human cartilage explants were treated with equipotent concentrations of named drugs to determine cell viability applying fluorescence microscopy., Results: Chondrotoxic effects increased from ropivacaine to mepivacaine to bupivacaine in a time-dependent and concentration-dependent manner. Compared with control, bupivacaine 0.5% decreased chondrocyte viability to 78% ± 9% (P = 0.0183) 1 hour and 16% ± 10% (P < 0.0001) 24 hours later, as determined by live-dead staining in monolayer cultures. Viability rates were reduced to 80% ± 7% (P = 0.0475) 1 hour and 80% ± 10% (P = 0.0095) 24 hours after treatment with ropivacaine 0.75%. After exposure to mepivacaine 2%, viable cells were scored 36% ± 6% (P < 0.0001) after 1 hour and 30% ± 11% (P < 0.0001) after 24 hours. Ropivacaine treatment was less chondrotoxic than bupivacaine (P = 0.0006) and mepivacaine exposure (P = 0.0059). Exposure to concentrations up to 0.25% of bupivacaine, 0.5% of ropivacaine, and 0.5% of mepivacaine did not reveal significant chondrotoxicity in flow cytometry. However, chondrotoxicity did not correlate with potency of local anesthetics. Immediate cell death was mainly due to necrosis followed by apoptosis. Cellular death rates were clearly higher in osteoarthritic compared with intact cartilage after bupivacaine, mepivacaine, and ropivacaine treatment in a decreasing order., Conclusion: Bupivacaine, ropivacaine, and mepivacaine are chondrotoxic in a time-dependent, concentration-dependent, and drug-dependent manner. Chondrotoxic and analgesic potencies do not directly correlate. Cellular death rates were higher in osteoarthritic compared with intact cartilage after local anesthetic treatment.

Purpose: Mesenchymal stem cells (MSCs) express markers of hypertrophic chondrocytes during chondrogenic differentiation. We tested the suitability of parathyroid hormone-related protein (PTHrP), a regulator of chondrocyte hypertrophy in embryonic cartilage development, for the suppression of hypertrophy in an in vitro hypertrophy model of chondrifying MSCs., Methods: Chondrogenesis was induced in human MSCs in pellet culture for two weeks and for an additional two weeks cultures were either maintained in standard chondrogenic medium or transferred to a hypertrophy-enhancing medium. PTHrP(1-40) was added to the medium throughout the culture period at concentrations from 1 to 1,000 pM. Pellets were harvested on days one, 14 and 28 for biochemical and histological analysis., Results: Hypertrophic medium clearly enhanced the hypertrophic phenotype, with increased cell size, and strong alkaline phosphatase (ALP) and type X collagen staining. In chondrogenic medium, 1-100 pM PTHrP(1-40) did not inhibit chondrogenic differentiation, whereas 1,000 pM PTHrP(1-40) significantly reduced chondrogenesis. ALP activity was dose-dependently reduced by PTHrP(1-40) at 10-1,000 pM in chondrogenic conditions. Under hypertrophy-enhancing conditions, PTHrP(1-40) did not inhibit the induction of the hypertrophy. At the highest concentration (1,000 pM) in the hypertrophic group, aggregates were partially dedifferentiated and differentiated areas of these aggregates maintained their hypertrophic appearance., Conclusions: PTHrP(1-40) treatment dose-dependently reduced ALP expression in MSC pellets cultured under standard chondrogenic conditions and is thus beneficial for the maintenance of the chondrogenic phenotype in this medium condition. When cultured under hypertrophy-enhancing conditions, PTHrP(1-40) could not diminish the induced enhancement of hypertrophy in the MSC pellets.

Purpose: Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-β or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis., Methods: We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 μg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium., Results: Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 μg/ml. An insulin concentration of 50 μg/ml had no additional effect compared with 10 μg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis., Conclusions: Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro.

Different approaches of engineering cartilage to treat defects in the articulating surfaces of the joints have been designed, which mainly use mesenchymal stem cells or autologous chondrocytes for in situ transplantation. However, these cells are poorly characterized with respect to viability, degree of differentiation and morphology or production of extracellular matrix. At present, one of the key approaches to generate chondrocytes is the stimulation of stem cells with transforming growth factor (TGF) β1. To characterize the molecular alterations occurring during the cellular transformation induced by TGF-β1 exposure, the differentiation process of bone marrow-derived stem cells into chondrocytes was investigated using an in vitro chondrogenesis model and the RNA arbitrarily primed PCR (RAP-PCR) fingerprinting technique. Distinct genes were found to be differentially regulated during chondrocyte development beginning on day 1: collagen type I, non-muscle myosin MYH9, followed by manganese superoxide dismutase and sodium-potassium ATPase on day 7. The results suggest that using RAP-PCR for differential display fingerprinting is a useful tool to investigate the differentiation process of bone marrow-derived stem cells following TGF-β1-stimulation.

Mesenchymal stromal cells have the potential to differentiate into a variety of mesenchymal tissues such as bone, cartilage and ligaments. The potential for the regeneration of bone with cartilage coverage has still not been achieved. We evaluated the ability of bone marrow mesenchymal stromal cells to regenerate osteochondral defects in the cavity of the lunate in an animal model. Autologous mesenchymal stromal cells were harvested from the iliac crest of New Zealand white rabbits and expanded in vitro. Total lunate excision was performed in 24 animals and the isolated cells were loaded onto scaffolds. Cell-free scaffolds were implanted in the lunate space of the right wrists of all animals, and the left lunate spaces were filled with predifferentiated, cell-loaded scaffolds. Radiographic and histological analyses were performed after two, six and 12 weeks. In addition, the animals were injected with a fluorescent agent every five days, starting at day 30. After two and six weeks there was no radiographic evidence of ossification, whereas after 12 weeks all animals showed radiographic evidence of ossification. Histological sections showed increasing evidence of cartilage-like cell formation at the edges and new bone tissue in the centre of the newly formed tissue in all groups. The histological examinations showed that bone tissue was located around the newly incorporated vascularisation. This study demonstrated that newly formed vascularisation is necessary for the regeneration of bone tissue with cell-loaded scaffolds.

Objective: We undertook this study to examine the effects of estradiol on chondrogenesis of human bone marrow-derived mesenchymal stem cells (MSCs), with consideration of sex-dependent differences in cartilage repair., Methods: Bone marrow was obtained from the iliac crest of young men. Density-gradient centrifugation-separated human MSCs proliferated as a monolayer in serum-containing medium. After confluence was achieved, aggregates were created and cultured in a serum-free differentiation medium. We added different concentrations of 17beta-estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane-impermeable E2-bovine serum albumin (E2-BSA), ICI 182.780 alone, G-1 (an agonist of G protein-coupled receptor 30 [GPR-30]), and G15 (a GPR-30 antagonist). After 21 days, the aggregates were analyzed histologically and immunohistochemically; we quantified synthesized type II collagen, DNA content, sulfated glycosaminoglycan (sGAG) concentrations, and type X collagen and matrix metalloproteinase 13 (MMP-13) expression., Results: The existence of intracellular and membrane-associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2-BSA in a dose-dependent manner. Furthermore, E2 enhanced type X collagen and MMP-13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR-30 agonists alone (G-1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G-1. Furthermore, the specific GPR-30 antagonist G15 reversed the GPR-30-mediated inhibition of chondrogenesis and up-regulation of hypertrophic gene expression., Conclusion: The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR-30). The study opens new perspectives for influencing chondrogenesis on the basis of classic and nonclassic estradiol signaling.

Recent investigations have shown the importance of scaffold pore size on the realisation of tissue engineered cartilage which promotes cell adhesion, proliferation and differentiation. The objective of this study was to investigate the influence of pore size on the mechanical properties, the permeability and the porosity of hyaluronan-collagen scaffolds. Hyaluronan-collagen scaffolds with three different mean pore sizes (302.5, 402.5 and 525 microm) have been produced according to a standardised protocol. The maximum stress at rupture, the Young's Moduli, permeability and porosity of the scaffolds were investigated. The permeability was determined both empirically and mathematically. Increased pore sizes indicated a larger stress at rupture as well as increased Young's Moduli. Porosity and permeability were raised by increasing pore sizes. The mathematically calculated permeability showed the same trend. The results indicate a higher mechanical stability for scaffolds with larger pores. The experimental and mathematical experiments both show increased permeability and fluid mobility for larger pores in scaffolds. Morphological changes resulting from the alteration of pore size led to non-correlation between the calculated and the experimental permeability.

Physical and chemical properties of the surfaces of implants are of considerable interest for dental and orthopedic applications. We used self-assembled monolayers (SAMs) terminated by various functional chemical groups to study the effect of surface chemistry on cell behavior. Cell morphology and proliferation on silicon wafers of various roughnesses and topographies created by chemical etching in caustic solution and by corundum sandblasting were analyzed as well. Water contact angle data indicated that oxidized wafer surfaces displayed high hydrophilicity, modification with poly(ethylene glycol) (PEG) created a hydrophilic surface, and an amino group (NH2) led to a moderately wettable surface. A hydrophobic surface was formed by hydrocarbon chains terminated by CH3, but this hydrophobicity was even further increased by a fluorocarbon (CF3) group. Cell proliferation on these surfaces was different depending primarily on the chemistry of the terminating groups rather than on wettability. Cell proliferation on CH3 was as high as on NH2 and hydrophilic oxidized surfaces, but significantly lower on CF3. Precoating of silicon wafers with cell culture serum had no significant influence on cell proliferation. Scanning electron microscopy indicated a very weak initial cell-surface contact on CF3. The cell number of osteoblasts was significantly lower on sandblasted surfaces compared with other rough surfaces but no differences were detected with 3T3 mouse fibroblasts. The different surface roughnesses and topographies were recognized by MG-63 osteoblasts. The cells spread well on smooth surfaces but appeared smaller on a rough and unique pyramid-shaped surface and on a rough sandblasted surface.

Collagen type-I is a major component of the extracellular matrix of most tissues and it is increasingly utilized for surface engineering of biomaterials to accelerate receptor-mediated cell adhesion. In the present study, coatings with layers of fibrillar type-I collagen were prepared on titanium, titanium alloy, and cobalt alloy to improve initial osteoblast adhesion and implant-tissue integration. To suppress the quick in vivo degradation rate of collagen the deposited layers were covalently immobilized at the metal surfaces as well as chemically cross-linked. The application of different oxidation techniques to the metallic substrates resulted in surfaces with varying hydroxyl group contents, which directly influenced the amount of immobilized silane coupling agents. It was found that a high density of surface-bound coupling agents increased the stability of the covalently linked collagen layers. After coating of metallic biomaterials with a cross-linked collagen layer, an improved cellular response of human osteoblast-like cells (MG-63) in vitro could be recognized.

It was shown recently that the deposition of thin films of tantalum and tantalum oxide enhanced the long-term biocompatibility of stainless steel biomaterials due to an increase in their corrosion resistance. In this study, we used this tantalum oxide coating as a basis for covalent immobilization of a collagen layer, which should result in a further improvement of implant tissue integration. Because of the high degradation rate of natural collagen in vivo, covalent immobilization as well as carbodiimide induced cross-linking of the protein was performed. It was found that the combination of the silane-coupling agent aminopropyl triethoxysilane and the linker molecule N,N'-disulphosuccinimidyl suberate was a very effective system for collagen immobilizing. Mechanical and enzymatic stability testing revealed a higher stability of covalent bound collagen layers compared to physically adsorbed collagen layers. The biological response induced by the surface modifications was evaluated by in vitro cell culture with human mesenchymal stem cells as well as by in vivo subcutaneous implantation into nude mice. The presence of collagen clearly improved the cytocompatibility of the stainless steel implants which, nevertheless, significantly depended on the cross-linking degree of the collagen layer.

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the species-related differences of collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Resistance against collagenase digestion, swelling ratio, amino acid sequence, shrinkage temperature, ultrastructural matrix morphology, crosslinking density and stress-strain characteristics were determined to evaluate the physico-chemical properties of equine- and bovine-collagen-based scaffolds. Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and time (p=0.0001) on degradation of the collagen samples by collagenase treatment. Crosslinked equine collagen samples showed a significantly reduced swelling ratio compared to bovine collagen samples (p< 0.0001). The amino acid composition of equine collagen revealed a higher amount of hydroxylysine and lysine. Shrinkage temperatures of non-crosslinked samples showed a significant difference between equine (60 degrees C) and bovine collagen (57 degrees C). Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and matrix condition (p=0.0001) on rupture strength measured by stress-strain analysis. The ultrastructure, the crosslinking density and the strain at rupture between collagen matrices of both species showed no significant differences. For tissue engineering purposes, the higher enzymatic stability, the higher form stability, as well as the lower risk of transmissible disease make the case for considering equine-based collagen. This study also indicates that results obtained for scaffolds based on a certain collagen species may not be transferable to scaffolds based on another, because of the differing physico-chemical properties.