Restoration a joint's articular surface following degenerative or traumatic pathology to the osteochondral unit pose a significant challenge. Recent advances have shown the utility of collagen-based scaffolds in the regeneration of osteochondral tissue. To provide these collagen scaffolds with the appropriate superstructure novel techniques in 3D printing have been investigated. This study investigates the use of polyɛ-caprolactone (PCL) collagen scaffolds in a porcine cadaveric model to establish the stability of the biomaterial once implanted. This study was performed in a porcine cadaveric knee model. 8mm defects were created in the medial femoral trochlea and repaired with a PCL collagen scaffold. Scaffolds were secured by one of three designs; Press Fit (PF), Press Fit with Rings (PFR), Press Fit with Fibrin Glue (PFFG). Mobilisation was simulated by mounting the pig legs on a continuous passive motion (CPM) machine for either 50 or 500 cycles. Biomechanical tensile testing was performed to examine the force required to displace the scaffold. 18 legs were used (6 PF, 6 PFR, 6 PFFG). Fixation remained intact in 17 of the cohort (94%). None of the PF or PFFG scaffolds displaced after CPM cycling. Mean peak forces required to displace the scaffold were highest in the PFFG group (3.173 Newtons, Standard deviation = 1.392N). The lowest peak forces were observed in the PFR group (0.871N, SD = 0.412N), while mean peak force observed in the PF group was 2.436N (SD = 0.768). There was a significant difference between PFFG and PFR (p = 0.005). There was no statistical significance in the relationship between the other groups. PCL reinforcement of collagen scaffolds provide an innovative solution for improving stiffness of the construct, allowing easier handling for the surgeon. Increasing the stiffness of the scaffold also allows press fit solutions for reliable fixation. Press fit PCL collagen scaffolds with and without fibrin glue provide dependable stability. Tensile testing provides an objective analysis of scaffold fixation. Further investigation of PCL collagen scaffolds in a live animal model to establish quality of osteochondral tissue regeneration are required

It is not known if the radiation sterilisation dose (RSD) of 25 kGy affects mechanical properties and biocompability of allograft bone by alteration of collagen triple helix or cross-links. Our aim was to investigate the mechanical and biological performance, cross-links and degraded collagen content of irradiated bone allografts. Human femoral shafts were sectioned into cortical bone beams (40 × 4 × 2 mm) and irradiated at 0, 5, 10, 15, 20, and 25 kGy for three-point bending tests. Corresponding cortical bone slices were used for in vitro determination of macrophage activation, osteoblast proliferation and attachment, and osteoclast formation and fusion. Subsequently, irradiated cortical bone samples were hydrolised for determination of pyridinoline (PYD), deoxypyridinoline (DPD), and pentosidine (PEN) by high performance liquid chromatography (HPLC) and collagen degradation by the alpha chymotrypsin (ï. j. CT) method. Irradiation up to 25 kGy did not affect the elastic properties of cortical bone, but the modulus of toughness was decreased from 87% to 74% of controls when the gamma dose increased from 15 to 25 kGy. Macrophages activation, the proliferation and attachment of osteoblasts on irradiated bone was not affected. Osteoclast formation and fusion were less than 40% of controls when cultured on bone irradiated at 25 kGy, and 80% at 15 kGy. Increasing radiation dose did not significantly alter the content of PYR, DPD or PEN but increased the content of denatured collagen. Cortical allografts fragility increases at doses above 15 kGy. Decreased osteoclast viability at these doses suggests a reduction in the capacity for bone remodelling. These changes were not correlated with alterations in collagen cross-links but in degradation to the collagen secondary structure as evidenced by increased content of denatured collagen

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of culture time. Although the characteristic anisotropy of tendon tissue is commonly replicated in scaffolds, relevant physical cues such as tendon rigidity or mechanical loading are often neglected. The objective of this study is to use tendons' main extracellular matrix component, collagen type I, to create scaffolds with an anisotropic surface topography and controlled rigidity, in an effort to engineer functional tendon tissue equivalents, with native organization and strength. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 μm) or planar polydimethylsiloxane (PDMS) molds and dried in a laminar flow hood to obtain 5 mg/ml collagen films. Surface topography and elastic modulus of the final scaffolds were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were isolated from adult tendon tissue and cultured on micro-grooved/planar scaffolds for 4, 7 and 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP at low pH and temperature. The elastic modulus of the scaffolds cross-linked with the highest concentration of 4SP matched the physiological values reported in developing tendons (∼15 kPa). Around eighty percent of the human tendon cells cultured on the cross-linked collagen films aligned in the direction of the anisotropy for 10 days in culture. At 4 days, tenoyctes cultured on micro-grooved substrates presented a significant higher nuclei aspect ratio than tenocytes cultured on planar substrates for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively in the rigid micro-grooved scaffolds after 7 days in culture. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities. Gene expression analysis and a replicate study with hBMSCs will be carried out to support the first results and to further identify the optimal biophysical conditions for tenogenic cell lineage commitment. This potentially leads to the design of smart implants that not only restore immediate tendon functionality but also provide microscopic cues that drive cellular synthesis of organized tissue-specific matrix

Treatment of segmental bone loss remains a major challenge in orthopaedic surgery. This study evaluated the healing potential of a series of highly porous tissue engineering scaffolds with the current clinical gold standard. We compare healing of collagen-glycosaminoglycan (CG) and collagen micro-hydroxyapatite (CHA) scaffolds, with and without recombinant bone morphogenetic protein-2 (BMP2), with autogenous bone graft (ABG) in the healing of a 15mm rabbit radius defect, which were filled with either CG scaffold, CHA scaffold, CG-BMP2, CHA-BMP2 or ABG. Serial radiographs and micro-computed tomography (µCT) at six week radiographs demonstrated complete defect bridging with callus using CHA and CG-BMP2 while the CHA-BMP2 was already in an advanced state of healing with cortical remodeling. By sixteen weeks CHA, CG-BMP2 and ABG all had advanced healing with cortical remodeling while CHA-BMP2 had complete anatomic healing. Quantitative histomorphometry values demonstrated similarly high healing levels of healing in CHA, CG-BMP2 and ABG with highest overall values in the CHA-BMP2 group. Thus, treatment of a critical sized, weight bearing, rabbit radius defect with a CHA scaffold can result in full cortical bridging with medullary cavity development. In addition, a CHA-BMP2 combination can result in fully mature, anatomic healing. The use of an off-the-shelf CHA scaffold for direct surgical placement into a defect site may be an effective bone graft substitute in the treatment of skeletal defects. The ease of manufacture, storage and peri-operative preparation may offer an alternative to traditional strategies, as well as to more recent BMP2 devices. This study provides clear evidence that CHA scaffolds can perform as well as autogenous bone grafts and supports their use as a viable alternative. Where the use of BMP2 may be desirable, these materials provide an ideal delivery mechanism and using a very low (near physiological) dose, healing superior to autogenous graft may be achieved

Introduction. Massive rotator cuff repairs have up to 60% failure rate and repair of a chronic repair can have up to 40% failure rate. With this in mind, new methodologies are being to being developed to overcome this problem. The use of tendon augmentation grafts is one of them. Prior attempts have shown equivocal or poorer outcomes to control repairs. Aims and objectives: The specific aim of these expereiments was to test how well ovine tendon cells would take to a specific biological augmentation graft (Ligamimetic), and wheter tissue engineering techniques would enhance this. Method. Tendon cells harvested from ovine tendons will be cultured, exposed to the tendon augmentation graft, and analysed to see how well it takes to the tendon cells. We have conducted a 21 day experiment, sampling at days 7, 14, and 21. The experiment will look in sheep tendon cells:1. Platelet rich plasma: A comparison of the effects of platelet rich plasma to cell adherence, cell proliferation, and collagen production. Mesenchymal stem cell: A comparison of the effects of mesenchymal stem cells to the material on cell adherence, cell proliferation, and collagen production. Results. Our results show that the ovine tendon cells do take to the tendon augmentation graft, and are able to proliferate. We will present DAPI stainings, DNA and collagen turnover, with Westin blotting results. Results show that the addition of PRP had increased the cell adherence, cell proliferation, and collagen production. These effects were not seen with the mesenchymal stem cells. Conclusion. These experiments have shown our novel collagen scaffold augmentation graft has allowed cells to proliferate on it. Tissue engineered techniques also enhance cell proliferation, and has the potential to enhance cell repair

Osteoarthritis (OA) is a multifactorial debilitating disease that affects over four million Canadians. Although the mechanism(s) of OA onset is unclear, the biological outcome is cartilage degradation. Cartilage degradation is typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II) – partly due to the up-regulation of catabolic enzymes - aggrecanases a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). There is currently no treatment that will prevent or repair joint damage, and current medications are aimed mostly at pain management. When pain becomes unmanageable arthroplastic surgery is often performed. Interest has developed over the presence of calcium crystals in the synovial fluid of OA patients, as they have been shown to activate synovial fibroblasts inducing the expression of catabolic agents. We recently discovered elevated levels of free calcium in the synovial fluid of OA patients and raised the question on its role in cartilage degeneration. Articular cartilage was isolated from 5 donors undergoing total hip replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase and expanded in DMEM supplemented with 10% heat-inactivated FBS. OA and normal human articular chondrocytes (PromoCell, Heidelberg, Germany) were transferred to 6-well plates in culture medium containing various concentrations of calcium (0.5, 1, 2.5, and 5 mM CaCl2), and IL-1β. Cartilage explants were prepared from the same donors and included cartilage with the cortical bone approximately 1 cm2 in dimension. Bovine articular cartilage explants (10 months) were used as a control. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. Immunohistochemistry was performed on cartilage explants to measure expression of Col X, MMP-13, and alkaline phosphatase. The sulfated glycosaminoglycan (GAG, predominantly aggrecan) content of cartilage was analyzed using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay, and aggregan fragmentation was determined by Western blotting using antibody targeted to its G1 domain. Western blotting was also performed on cell lysate from both OA and normal chondrocytes to measure aggrecan, Col II, MMP-3 and −13, ADAMTS-4 and −5. Ca2+ significantly decreased the proteoglycan content of the cartilage explants as determined by the DMMB assay. The presence of aggrecan and Col II also decreased as a function of calcium, in both the human OA and bovine cartilage explants. When normal and OA chondrocytes were cultured in medium supplemented with increasing concentrations of calcium (0.5–5 mM Ca2+), aggrecan and Col II expression decreased dose-dependently. Surprisingly, increasing Ca2+ did not induce the release of MMP-3, and −13, or ADAMTS-4 and-5 in conditioned media from OA and normal chondrocytes. Interestingly, inhibition of the extracellular calcium-sensing receptor CaSR) reversed the effects of calcium on matrix protein synthesis. We provide evidence that Ca2+ may play a direct role in cartilage degradation by regulating the expression of aggrecan and Col II through activation of CaSR

Introduction. Management of painful scarring of a nerve after surgery or trauma is challenging. Wrapping with a vein or adhesion barriers such as silicone have been reported with some success. This is a retrospective review of application of a xenograft collagen wrap around a peripheral nerve following neurolysis. There is no report in English literature of use of a collagen wrap for this purpose. Materials/Methods. 12 patients underwent use of the Neuragen™ bovine collagen nerve conduit or NeuraWrap™ (Integra Life Sciences, Plainsboro, NJ) over a 6 year period. The causes were, scarring after carpal tunnel (n=5) or cubital tunnel release (n=3) or partial nerve injury (n=4, radial, median, dorsal ulnar, digital nerves). Follow up ranged from 6–12 months. Outcome was assessed by VAS or functional improvement. Results. No patient developed adverse reaction to the xenograft conduit. All 10 patients with painful scarring reported improvement of pain with reduction in mean VAS from 7 to 2 at final follow-up. The patient with radial palsy with scarring of the nerve had full recovery of radial nerve function by 6 months. The partially injured sensory branch of median nerve and digital nerve regained normal 2PD. The patients with scarring after decompression surgery had improvement in pain and range of motion of adjacent joints but did not show any change in clinical neurological status. Conclusions. A collagen conduit wrapped around a partially injured or scarred nerve is safe and associated with clinical improvement in pain and nerve function

Purpose. Recent work has shown that joint contracture severity can be decreased with the mast cell stabilizer ketotifen in association with decreased numbers of myofibroblasts and mast cells in the joint capsule of a rabbit model of post-traumatic contractures. Neuropeptides such as Substance P (SP) can induce mast cells to release growth factors. Using a gel contraction assay, we test the hypothesis that joint capsule cell-mediated contraction of a collagen gel can be enhanced with SP, but the effect is magnified in the presence of mast cells. Method. Anterior elbow joint capsules were obtained at the time of surgical release from 2 men (age 34 and 54) and 1 woman (age 40) with chronic (> 1 year) post-traumatic joint contractures. The human mast cell line HMC-1 (Mayo Clinic, Rochester), SP and the NK1 receptor antagonist RP67580 (Sigma, Oakville, ON) were used. NK1 is the SP receptor. Neutralized Collagen solution composed with 58% Vitrogen 100 purified collagen mixed with HMC-1 cells only (7.5 105), human capsule cells (2.5 105), or human capsule cells (2.5 105) and 7.5 105 mast cells (1:3) were cast into 24- well tissue culture plates. In some experiments, SP (1 × 10. −5. M) +/− RP67580 (0.5 mM) were added. The gels were maintained with 0.5 ml DMEM composed with 2% BSA and incubated at 37C for 12 h for gelation to occur. The gels were then detached from the wall and the bottom of culture plate wells, and photographed at regular intervals up to 72 hours. Gel contraction studies were carried out on passage 4 and done in triplicate for each patient. The average value of each patients triplicate was combined to give a mean contraction at each time point. Statistical analysis involved an ANOVA with posthoc Bonferroni correction. P < 0.001 was significant. Results. Mast cells alone or with SP were unable to contract collagen gels. Joint capsule cells were able to contract the collagen gels and this was enhanced in the presence of SP, although not statistically significant. Joint capsule cells combined with mast cells enhanced the gel contraction more than joint capsule cells alone or with SP (p<0.001). The addition of SP accelerated the joint capsule cell-mediated gel contraction in the presence of mast cells the greatest (p<0.001 over all other conditions). The inhibitor RP67580 completely abolished the collagen gel contraction of the joint capsule cells in all conditions. Conclusion. The in vitro experiment shows that joint capsule cell function, in the form of collagen gel contraction, is modified by the presence of mast cells and neuropeptides. These findings are significant as they strengthen the hypothesis that a myofibroblast mast cell neuropeptide fibrosis axis may be contributing to the joint capsule changes underling the loss of motion in post-traumatic joint contractures. In vivo studies with the rabbit model of post-traumatic contractures will be performed using the compounds examined in the current study

Our previous study has revealed that silver nanoparticles (AgNPs) have potential to promote wound healing by accelerated re-epithelization and enhanced differentiation of fibroblasts. However, the effect of AgNPs on the functionality of repaired skin is unknown. The aim of this study was to explore the tensile properties of healed skin after treatment with AgNPs. Immunohistochemical staining, quantitative assay and scanning electron microscopy (SEM) were used to detect and compare collagen deposition, and the morphology and distribution of collagen fibers. Our results showed that AgNPs improved tensile properties and led to better fibril alignments in repaired skin, with a close resemblance to normal skin. Based on our findings, we concluded that AgNPs were predominantly responsible for regulating deposition of collagen and their use resulted in excellent alignment in the wound healing process. The exact signaling pathway by which AgNPs affect collagen regeneration is yet to be investigated

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. Quality of cartilaginous repair tissue following BMSC transplantation has been shown to correlate with functional outcome. Therefore, tissue-engineering variables, such as cell expansion environment and seeding density of scaffolds, are currently under investigation. The objectives of this study were to demonstrate chondrogenic differentiation of BMSCs seeded within a collagen I scaffold following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments, and assess the impact of seeding density on in vitro chondrogenesis. It was hypothesised that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 million cells/cm3. Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing expansion medium, and seeded within collagen I scaffolds (6 mm diameter, 3.5 mm thickness and 0.115 ± 0.020 mm pore size; Integra LifeSciences Corp.) at densities of 50, 10, 5, 1, and 0.5 million BMSCs/cm3. For 3D isolation and expansion, bone marrow aspirates containing known quantities of mononucleated cells (BMNCs) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 million BMNCs/cm3 and cultured in expansion medium for an equivalent duration to 2D expansion. All cell-scaffold constructs were differentiated in vitro in chondrogenic medium containing transforming growth factor-beta three for 21 days and assessed with RT-qPCR, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II mRNA relative to pre-differentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 million BMSCs/cm3. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 million BMSCs/cm3 based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/DNA. For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 million BMNCs/cm3, while collagen II deposition occurred in scaffolds seeded at 10–50 million BMNCs/cm3. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 million BMNCs/cm3. Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 million BMSCs/cm3 and 50 million BMNCs/cm3, respectively. Accordingly, these densities could be considered when seeding collagen I scaffolds in BMSC transplantation protocols

Purpose. Platelet-derived growth factor-BB (PDGF-BB) is a well characterized wound healing protein known to be chemotactic and mitogenic for cells of mesenchymal origin, including osteoblasts and chondrocytes. Biocompatible scaffolds, combined with growth factors such as PDGF-BB, have potential to stimulate regeneration and repair of osseous and cartilaginous tissues. The purpose of this study was to determine the efficacy and safety of recombinant human PDGF-BB (rhPDGF-BB) combined with a collagen implant to augment healing of osteochondral defects. Method. A single osteochondral defect (8mm x 8mm) was created in the medial femoral condyle of 32 adult goats. Collagen implants(8.5mm x 8mm) hydrated with four doses of rhPDGF-BB (0g, 15g, 75g, 500g) were press-fit into the defect. Defects in four animals were left untreated. All goats were sacrificed 12 weeks postoperatively. Macroscopic evaluation and quantitative CT analyses were performed. Histologic sections were stained with Safranin O/Fast Green and assessed with a modified ODriscoll scoring scale for cartilage and bone repair. Significance was determined by One-Way ANOVA or nonparametric Kruskal-Wallis. Results. Macroscopic evaluation indicated significant improvement of the gross cartilage repair score for the rhPDGF-BB treatment groups compared to the 0g rhPDGF-BB control (500g;0g) and empty defect groups (500,75,15g; Empty). MicroCT analysis indicated a significant increase in trabecular number for the 500g group compared to 0g control, 75g, and Empty groups(p=0.004). Average bone volume reconstitution for the 500g group was increased (58.8%) compared to the 0g control. The total cartilage repair score was significantly improved (p=0.048) in the 500g treatment group (14.30.3) compared to the 0g control group (12.10.4). All rhPDGF-BB treatment groups exhibited increased Safranin-O staining of the matrix compared to the 0g control group, and a significantly decreased incidence(p=0.01) of subchondral cyst formation compared to the empty defect group. Conclusion. The results of this study indicate that rhPDGF-BB, combined with a collagen implant, is safe and improves repair of large osteochondral defects located in a high-load bearing region in a caprine model. Increases in gross scoring and histopathologic cartilage repair score for the rhPDGF-BB treatment groups, in addition to the presence of bony bridging, especially for the 500g rhPDGF-BB treatment group, indicate enhanced reconstitution of the subchondral bone and overlying repair tissue. The cartilage repair score was increased, on average, in the empty defect group relative to the 0g rhPDGF-BB group, however this score may be partially inflated due to collapse of the surrounding native tissue into the defect. Combined with a significant decrease in cyst formation in all rhPDGF-BB treatment groups, these results suggest that rhPDGF-BB, combined with a collagen implant, may have promise as a therapeutic agent for osteochondral defect repair

Purpose. Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold. Method. Chondrocytes were isolated from Outerbridge grade 0 and 1 AC from four patients undergoing total knee arthroplasty and embedded within 216 bovine collagen I scaffolds. Scaffolds were incubated in hypoxic (3% O2) or normoxic (21% O2) conditions for 1hr, 21hr and 14 days. Gene expression was determined using Q-rt-PCR for col I/II/X, COMP, SOX9, aggrecan and B actin. Matrix production was determined using glycosaminoglycan (GAG) content relative to cell count determined by DNA quantification. Cell viability and location within the matrix was determined by Live/Dead assay and confocal microscopy. Statistical analysis was performed using a two-tailed T-test. Results. Chondrocytes cultured under hypoxic conditions showed an upregulation of all matrix related genes compared to normoxic conditions noted most markedly in col II, COMP and SOX9 expression. There were similar numbers of chondrocytes between hypoxic and normoxic groups (P=0.68) but the chondrocytes in the hypoxic group produced more GAG per cell (P= 0.052). Viable cells were seen throughout the matrix in both groups. Conclusion. Important matrix related genes (col II, COMP, SOX9) were most significantly upregulated in hypoxic conditions compared to normoxic conditions. This was supported by an increase in GAG production per cell in hypoxic conditions. The results indicate that hypoxia induces an upregulation in the production of extracellular matrix components typical of AC with only modest increases in col I (possibly related to the col I based scaffold used in this experiment). These results indicate that hypoxic conditions are important for the maintenance of chondrocyte phenotype even when the cells are cultured in a 3D environment. In conclusion, hypoxic culture conditions should be used to help maintain chondrocyte phenotype even when culturing these cells in a 3D scaffold

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Human ACL cells were harvested from ligament samples donated by patients who underwent total knee arthroplasties with informed consents. Interface cells were isolated from the 5-mm-end of ACL. Midsubstance cells were cultured from the middle part of ACL. The cells were seeded onto stretch chambers (2Ä−2 cm, 50,000 cells/chamber) and uni-axial cyclic mechanical stretch (0.5 Hz, 7%) was applied for 2 h using a ST140. RNA samples were reverse-transcripted and quantitative real-time RT-PCR analysis were performed. To inhibit the function of integrin alphaVbeta3 subunit or alpha5 in stretching experiments, anti-human integrin alphaVbeta3 and alpha5 functional blocking antibodies (alphaVbeta3: 20 mg/ml, alpha5: 4 mg/ml) were used. To investigate the cellular attachments responding to mechanical stretch, we observed the distribution of integrins and stress fibers in both ACL cells. The shape of midsubstance cells showed spindle and fibroblastic cellular morphologies. On the other hand, the interface cells displayed chondroblastic appearances such as small and triangular morphologies. The expressions of COL1A1, COL2A1, and COL3A1 genes were detected in the tissue RNAs of interface zones. However, these expressions were decreased in cultured interface cells. In midsubstance cells, the expression of COL1A1 gene was equally detected in both tissues and cultured cells. COL3A1 gene expression was maintained in cultured midsubstance cells. These results indicated that the phenotypes of both ACL cells were changed by cultured conditions, especially in the interface cells. After mechanical stretch, the COL1A1 expression of midsubstance and interface cells were stimulated up to 6 and 14-fold levels of each non-stretched control, respectively. The COL3A1 expressions were also enhanced up to 1.8-fold level of controls by stretching treatment in both cells. Integrin alphaVbeta3 was shifted to the peripheral edge of cells by stretching treatment. In addition, mechanical stretch changed the integrin alphaVbeta3-dependent stress fiber formation in both ACL cells. The functional blocking of integrin alphaVbeta3 inhibited stretch-activated COL1A1 and COL3A1 expressions. However, the functional blocking of integrin alpha5 did not suppress the stretch-induced COL1A1 and COL3A1 expressions in both ACL cells. Cultured interface cells loose their phenotypes in collagen gene expressions. However, mechanical stretch reproduces the expression of COL1A1 and COL3A1 genes in cultured ACL cells. The present study demonstrated that stretch-activated collagen gene expressions depend on the integrin alphaVbeta3-mediated cellular adhesions

The aim of our project is to develop resorbable nanostructured composite layer with controlled elution of antibiotics for implants survival rate enhancement. The nanostructured layers are expected to be used especially in the case of known systemic or local (joint) inflammation. This layer can provide a bone tissue/implant (titanium alloy) bioactive interface improving the physiological healing process and eliminating the risk of bacterial orthopedic infections. The main aim of this study was to verify whether the local concentration of released vancomycin exceeded the minimum inhibitory concentration (MIC) for vancomycin-resistant Staphylococcus aureus (VRSA, >16 mg/l). The layer is composed of collagen (type I, isolated form calf skin), hydroxyapatite nanoparticles and vancomycin hydrochloride (10 wt%). The stability of collagen was enhanced by EDC/NHS cross-linking. The in vitro release of vancomycin and crystalline degradation products from optimally cross-linked layers was investigated. An elution method and a high performance liquid chromatographic assay were employed to characterize the in vitro release rates of the vancomycin and its crystalline degradation antibacterial inactive products over a 21-day period. During the whole experimental period, the level of released vancomycin was high above the MIC for VRSA. The maximum average concentration was obtained between day 4 and day 8 and it reached 265 mg/l. At the end of the experiment (day 21), an average concentration of 104 mg/l was detected. Our study confirmed the prophylactic effects of studied vancomycin-loaded nanostructured layers

Purpose. A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results. Results. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N (55 vs. 78 % of control OA cells). Results also showed that the decreased type X collagen mRNA level was maintained not only when cells were removed from the PPE:N surfaces and transferred to new polystyrene culture dishes in the presence of chondrogenic media, but also when transferred to pellet cultures. Culturing MSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. Conclusion. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results may lead us one step closer to the production of large amounts of reprogrammed MSCs for tissue engineering applications

Calcification of the intervertebral disc (IVD) has been correlated with degenerative disc disease (DDD), a common cause of low back pain. The appearance of calcium deposits has been shown to increase with age, and its occurrence has been associated with several other disorders such as hyperparathyroidism, chondrocalcinosis, and arthritis. Trauma, vertebral fusion and infection have also been shown to increase the incidence of IVD calcification. The role of IVD calcification in the development DDD is unknown. Our preliminary data suggest that ionic calcium content and expression of the extracellular calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR) and regulator of calcium homeostasis, are increased in the degenerated discs. However, its role in DDD remains unclear. IVD Cells: Bovine and normal human IVD cells were incubated in PrimeGrowth culture medium (Wisent Bioproducts, Canada; Cat# 319–510-CL, −S1, and S2) and supplemented with various concentrations of calcium (1.0, 1.5, 2.5, 5.0 mM), a CaSR agonist [5 µM], or IL-1β [10 ng/ml] for 7 days. Accumulated matrix protein was quantitated for aggrecan and type II collagen (Col II) by Western blotting. Conditioned medium was also collected from cells treated for 24h and measured for the synthesis and release of total proteoglycan using the DMMB assay and Western blotting for Col II content. IVD Cultures: Caudal IVDs from tails of 20–24 month old steers were isolated with the PrimeGrowth Isolation kit (Wisent Bioproducts, Canada). IVDs were cultured for 4 weeks in PrimeGrowth culture medium supplemented with calcium (1.0, 2.5, or 5.0 mM), or a CaSR agonist [5 µM]. Cell viability was measured in NP and AF tissue using Live/Dead Imaging kit (ThermoFisher, Waltham, MA), to determine if Ca2+ effects cell viability end the expression of aggrecan and Col II was evaluated in the IVD tissue by Western blotting. Histological sections were prepared to determine total proteoglycan content, alkaline phosphatase expression and degree of mineralisation by von Kossa staining. The accumulation of aggrecan and Col II decreased dose-dependently in IVD cells following supplementation with calcium or the CaSR agonist. Conditioned medium also demonstrated decreases in the synthesis and release of proteoglycan and collagen with increasing Ca2+ dose or direct activation of the CaSR with agonist. A similar phenomenon was observed for total proteoglycan and aggrecan and Col II in IVDs following calcium supplementation or the CaSR agonist. In addition to decreases in Col II and aggrecan, increases in alkaline phosphatase expression and mineralisation was observed in IVDs cultured in elevated Ca2+ concentrations without affecting cell viability. Our results suggest that changes in the local concentrations of calcium are not benign, and that activation of the CaSR may be a contributing factor in IVD degeneration. Determining ways to minimise Ca2+ infiltration into the disc may mitigate disc degeneration

The results for autologous chondrocyte implantation (ACI) in the treatment of osteochondral defects in the knee are encouraging. At present, two techniques have been described to retain the chondrocyte suspension within the defect. The first involves using a periosteal flap harvested from the distal femur and the second involves using a type I/III collagen membrane. To the authors' knowledge there are no comparative studies of these two techniques in the current literature. A total of 68 patients with a mean age of 30.52 years (range 15 to 52 years) with symptomatic articular cartilage defects were randomised to have either ACI with a periosteal cover (33 patients) or ACI with a type I/III collagen cover (35 patients). The mean defect size was 4.54 cm. 2. (range 1 to 12 cm. 2. ). All patients were followed up at 24 months. A functional assessment using the Modified Cincinnati score showed that 74% of patients had a good or excellent result following the ACI with collagen cover compared with 67% after the ACI with periosteum cover at 2 years (p>0.05). Arthroscopy at 1 year also demonstrated similar results for both techniques. However, 36.4% of the periosteum covered grafts required shaving for hypertrophy compared with 1 patient for the collagen covered technique. This prospective, randomised study has shown no statistical difference between the clinical outcome of ACI with a periosteal cover versus ACI with a collagen cover at 2 years. A significant number of patients who had the ACI with periosteum technique required shaving of a hypertrophied graft within the first year of surgery. We conclude that there is no advantage in using periosteum as a cover for retaining the chondrocytes within an osteochondral defect; as a result we advocate the use of an alternative cover such as a porcine-derived, type I/III collagen membrane

This study investigated confocal laser scanning microscopy (CLSM) as a novel method of imaging of chondrocytes on a collagen membrane used for articular cartilage repair. Cell viability and the effects of surgery on the cells were assessed. Cell images were acquired under four conditions: 1, Pre-operative 2, After handling 3, Heavily grasped with forceps 4, Cut around the edge. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 1.12–1.68 ± 0.22 × 10. 6. cells/cm. 2. in specimens without significant trauma (n=25 images), this decreased to 0.253 × 10. 6. cells/cm. 2. in the specimens that had been grasped with forceps (p <0.001) (5 images). Cell viability on delivery grade membrane was 86.8±2.1%. The viability dropped to 76.3 ± 1.6% after handling and 35.1 ± 1.7% after crushing with forceps. Where the membrane was cut with scissors, there was a band of cell death where the viability dropped to 17.3 ± 2.0% compared to 73.4 ± 1.9% in the adjacent area (p <0.001). Higher magnification revealed cells did not have the rounded appearance of chondrocytes. CLSM can quantify and image the fine morphology of cells on a MACI membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery

Articular cartilage repair remains a challenge in orthopedic surgery, as none of the current clinical therapies can regenerate the functional hyaline cartilage tissue. In this study, we proposed a one-step surgery strategy that uses autologous bone marrow mesenchymal stem cells (MSCs) embedded in type II collagen (Col-II) gels to repair the full thickness chondral defects in minipig models. Briefly, 8 mm full thickness chondral defects were created in both knees separately, one knee received Col-II + MSCs transplantation, while the untreated knee served as control. At 1, 3 and 6 months postoperatively, the animals were sacrificed, regenerated tissue was evaluated by magnetic resonance imaging, macro- and microscopic observation, and histological analysis. Results showed that regenerated tissue in Col-II + MSCs transplantation group exhibited significantly better structure compared with that in control group, in terms of cell distribution, smoothness of surface, adjacent tissue integration, Col-II content, structure of calcified layer and subchondral bone. With the regeneration of hyaline-like cartilage tissue, this one step strategy has the potential to be translated into clinical application

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone