Rapid isolation of bone marrow mesenchymal stromal cells using integrated centrifuge-based technology.

BACKGROUND AIMS: The use of bone marrow-derived mesenchymal stromal cells (MSCs) in cell-based therapies is currently being developed for a number of diseases. Thus far, the clinical results have been inconclusive and variable, in part because of the variety of cell isolation procedures and culture conditions used in each study. A new isolation technique that streamlines the method of concentration and demands less time and attention could provide clinical and economic advantages compared with current methodologies. In this study, we evaluated the concentrating capability of an integrated centrifuge-based technology compared with standard Ficoll isolation. METHODS: MSCs were concentrated from bone marrow aspirate using the new device and the Ficoll method. The isolation capabilities of the device and the growth characteristics, secretome production, and differentiation capacity of the derived cells were determined. RESULTS: The new MSC isolation device concentrated the bone marrow in 90 seconds and resulted in a mononuclear cell yield 10-fold higher and with a twofold increase in cell retention compared with Ficoll. The cells isolated using the device were shown to exhibit similar morphology and functional activity as assessed by growth curves and secretome production compared to the Ficoll-isolated cells. The surface marker and trilineage differentiation profile of the device-isolated cells was consistent with the known profile of MSCs. DISCUSSION: The faster time to isolation and greater cell yield of the integrated centrifuge-based technology may make this an improved approach for MSC isolation from bone marrow aspirates.

Decellularized extracellular matrix microparticles seeded with bone marrow mesenchymal stromal cells for the treatment of full-thickness cutaneous wounds.

Extracellular matrix materials mechanically dissociated into submillimeter particles have a larger surface area than sheet materials and enhanced cellular attachment. Decellularized porcine mesothelial extracellular matrix microparticles were seeded with bone marrow-derived mesenchymal stromal cells and cultured in a rotating bioreactor. The mesenchymal stromal cells attached and grew to confluency on the microparticles. The cell-seeded microparticles were then encapsulated in varying concentrations of fibrin glue, and the cells migrated rapidly off the microparticles. The combination of microparticles and mesenchymal stromal cells was then applied to a splinted full-thickness cutaneous in vivo wound model. There was evidence of increased cell infiltration and collagen deposition in mesenchymal stromal cells-treated wounds. Cell-seeded microparticles have potential as a cell delivery and paracrine therapy in impaired healing environments.

Two-Year Evaluation of Osteochondral Repair with a Novel Biphasic Graft Saturated in Bone Marrow in an Equine Model.

Objective To evaluate a biphasic cartilage repair device (CRD) for feasibility of arthroscopic implantation, safety, biocompatibility, and efficacy for long-term repair of large osteochondral defects. Methods The CRD was press-fit into defects (10 mm diameter, 10 mm deep) created in the femoral trochlea of 12 horses. In the contralateral limb, 10 mm diameter full-thickness chondral defects were treated with microfracture (MFX). Radiographs were obtained pre- and postoperatively, and at 4, 12, and 24 months. Repeat arthroscopy was performed at 4 and 12 months. Gross assessment, histology, mechanical testing, and magnetic resonance imaging (MRI) were performed at 24 months. Results The CRD was easily placed arthroscopically. There was no evidence of joint infection, inflammation, or degeneration. CRD-treated defects had significantly more sclerosis compared to MFX early ( P = 0.0006), but was not different at 24 months. CRD had better arthroscopic scores at 4 months compared to MFX ( P = 0.0069). At 24 months, there was no difference in repair tissue on histology or mechanical testing. Based on MRI, CRD repair tissue had less proteoglycan (deep P = 0.027, superficial P = 0.015) and less organized collagen (deep P = 0.028) compared to MFX. Cartilage surrounding MFX defects had more fissures compared to CRD. Conclusion The repair tissue formed after CRD treatment of a large osteochondral lesion is fibrocartilage similar to that formed in simple chondral defects treated with MFX. The CRD can be easily placed arthroscopically, is safe, and biocompatible for 24 months. The CRD results in improved early arthroscopic repair scores and may limit fissure formation in adjacent cartilage.

Effects of mechanical stimulation on the biomechanics and histology of stem cell-collagen sponge constructs for rabbit patellar tendon repair.

The objective of this study was to determine how mechanical stimulation affects the biomechanics and histology of stem cell-collagen sponge constructs used to repair central rabbit patellar tendon defects. Autogenous tissue-engineered constructs were created for both in vitro and in vivo analyses by seeding mesenchymal stem cells from 10 adult rabbits at 0.14x10(6) cells/construct in type I collagen sponges. Half of these constructs were mechanically stimulated once every 5 min for 8 h/day to a peak strain of 4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. Samples allocated for in vitro testing revealed that mechanically stimulated constructs had 2.5 times the linear stiffness of nonstimulated constructs. The remaining paired constructs for in vivo studies were implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Twelve weeks after surgery, repair tissues were assigned for biomechanical (7 pairs) and histologic (3 pairs) analyses. Maximum force, linear stiffness, maximum stress, and linear modulus for the stimulated (vs. nonstimulated) repairs averaged 70% (vs. 55%), 85% (vs. 55%), 70% (vs. 50%), and 50% (vs. 40%) of corresponding values for the normal central third of the patellar tendons. The average force-elongation curve for the mechanically stimulated repairs also matched the corresponding curve for the normal patellar tendons, up to 150% of the peak in vivo force values recorded in a previous study. Construct and repair linear stiffness and linear modulus were also positively correlated (r = 0.6 and 0.7, respectively). Histologically both repairs showed excellent cellular alignment and mild staining for decorin and collagen type V, and moderate staining for fibronectin and collagen type III. This study shows that mechanical stimulation of stem cell-collagen sponge constructs can significantly improve tendon repair biomechanics up to and well beyond the functional limits of in vivo loading.

Osteoblastogenesis of Mesenchymal Stem Cells in 3-D Culture Enhanced by Low-Intensity Pulsed Ultrasound through Soluble Receptor Activator of Nuclear Factor Kappa B Ligand.

This study was performed to investigate osteoblastogenesis of human mesenchymal stem cells (hMSCs) cultured in 3-D scaffolds stimulated with low-intensity pulsed ultrasound and to identify the underlying mechanism mediated by soluble receptor activator of nuclear factor kappa B ligand (sRANKL) secreted by hMSCs. The results indicate that the mRNA levels of core-binding factor subunit alpha subunit 1 (CBFA1), osterix (OSX), alkaline phosphatase (ALP), osteocalcin and osteoprotegerin (OPG) and sRANKL production of hMSCs stimulated by ultrasound were significantly increased compared with the levels without ultrasound stimulation. Attenuating the sRANKL activity of ultrasound-treated hMSCs significantly reduced the mRNA expression of CBFA1, OSX, ALP and OPG. Adding sRANKL in hMSC culture significantly increased the mRNA expression of CBFA1, OSX and OPG. Together, the results suggest that osteoblastogenesis of hMSCs enhanced by ultrasound stimulation is mediated by endogenous sRANKL.

Do changes in the mechanical properties of articular cartilage promote catabolic destruction of cartilage and osteoarthritis?

Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration, a thickening of subchondral bone, and formation of marginal osteophytes. Previous mechanical characterization of cartilage in our laboratory suggests that energy storage and dissipation is reduced in osteoarthritis as the extent of fibrillation and fissure formation increases. It is not clear whether the loss of energy storage and dissipation characteristics is a result of biochemical and/or biophysical changes that occur to hyaline cartilage in joints. The purpose of this study is to present data, on the strain rate dependence of the elastic and viscous behaviors of cartilage, in order to further characterize changes that occur in the mechanical properties that are associated with OA. We have previously hypothesized that the changes seen in the mechanical properties of cartilage may be due to altered mechanochemical transduction by chondrocytes. Results of incremental tensile stress-strain tests at strain rates between 100%/min and 10,000%/min conducted on OA cartilage indicate that the slope of the elastic stress-strain curve increases with increasing strain rate, unlike the reported behavior of skin and self-assembled collagen fibers. It is suggested that the strain-rate dependence of the elastic stress-strain curve is due to the presence of large quantities of proteoglycans (PGs), which protect articular cartilage by increasing the apparent stiffness. The increased apparent stiffness of articular cartilage at high strain rates may limit the stresses borne and prolong the onset of OA. It is further hypothesized that increased compressive loading of chondrocytes in the intermediate zone of articular cartilage occurs as a result of normal wear to the superficial zone or from excessive impact loading. Once the superficial zone of articular cartilage is worn away, the tension is decreased throughout all cartilage zones leading to increased chondrocyte compressive loading and up-regulation of mechanochemical transduction processes that elaborate catabolic enzymes.

Elastic energy storage in human articular cartilage: estimation of the elastic modulus for type II collagen and changes associated with osteoarthritis.

The viscoelastic mechanical properties of normal and osteoarthritic articular were analyzed based on data reported by Kempson [in: Adult Articular Cartilage (1973)] and Silver et al. (Connect. Tissue Res., 2001b). Results of the analysis of tensile elastic stress-strain curves suggest that the elastic modulus of cartilage from the superficial zone is approximately 7.0 GPa parallel and 2.21 GPa perpendicular to the cleavage line pattern. Collagen fibril lengths in the superficial zone were found to be approximately 1265 microm parallel and 668 microm perpendicular to the cleavage line direction. The values for the elastic modulus and fibril lengths decreased with increased extent of osteoarthritis. The elastic modulus of type II collagen parallel to the cleavage line pattern in the superficial zone approaches that of type I collagen in tendon, suggesting that elastic energy storage occurs in the superficial zone due to the tensile pre-tension that exists in this region. Decreases in the elastic modulus associated with osteoarthritis reflect decreased ability of cartilage to store elastic energy, which leads to cartilage fibrillation and fissure formation. We hypothesize that under normal physiological conditions, collagen fibrils in cartilage function to store elastic energy associated with weight bearing and locomotion. Enzymatic cleavage of cartilage proteoglycans and collagen observed in osteoarthritis may lead to fibrillation and fissure formation as a result of impaired energy storage capability of cartilage.

Mechanobiology of cartilage: how do internal and external stresses affect mechanochemical transduction and elastic energy storage?

Articular cartilage is a multilayered structure that lines the surfaces of all articulating joints. It contains cells, collagen fibrils, and proteoglycans with compositions that vary from the surface layer to the layer in contact with bone. It is composed of several zones that vary in structure, composition, and mechanical properties. In this paper we analyze the structure of the extracellular matrix found in articular cartilage in an effort to relate it to the ability of cartilage to store, transmit, and dissipate mechanical energy during locomotion. Energy storage and dissipation is related to possible mechanisms of mechanochemical transduction and to changes in cartilage structure and function that occur in osteoarthritis. In addition, we analyze how passive and active internal stresses affect mechanochemical transduction in cartilage, and how this may affect cartilage behavior in health and disease.

Comparison of Three Methods to Quantify Repair Cartilage Collagen Orientation.

OBJECTIVE: The aim of this study was to determine if the noninvasive or minimally invasive and nondestructive imaging techniques of quantitative T2-mapping or multiphoton microscopy (MPM) respectively, could detect differences in cartilage collagen orientation similar to polarized light microscopy (PLM). It was hypothesized that MRI, MPM, and PLM would all detect quantitative differences between repair and normal cartilage tissue. METHODS: Osteochondral defects in the medial femoral condyle were created and repaired in 5 mature goats. Postmortem, MRI with T2-mapping and histology were performed. T2 maps were generated and a mean T2 value was calculated for each region of interest. Histologic slides were assessed using MPM with measurements of autocorrelation ellipticity, and by PLM with application of a validated scoring method. Collagen orientation using each of the 3 modalities (T2-mapping, MPM, and PLM) was measured in the center of the repair tissue and compared to remote, normal cartilage. RESULTS: MRI, MPM, and PLM were able to detect a significant difference between repair and normal cartilage (n = 5). The average T2 value was longer for repair tissue (41.43 ± 9.81 ms) compared with normal cartilage (27.12 ± 14.22 ms; P = 0.04); MPM autocorrelation ellipticity was higher in fibrous tissue (3.75 ± 1.17) compared with normal cartilage (2.24 ± 0.51; P = 0.01); the average PLM score for repair tissue was lower (1.6 ± 1.02) than the score for remote normal cartilage (4.4 ± 0.42; P = 0.002). The strongest correlation among the methods was between MRI and PLM (r = -0.76; P = 0.01), followed by MPM and PLM (r = -0.58; P = 0.08), with the weakest correlation shown between MRI and MPM (r = 0.35; P = 0.31). CONCLUSION: All 3 imaging methods quantitatively measured differences in collagen orientation between repair and normal cartilage, but at very different levels of resolution. PLM is destructive to tissue and requires euthanasia, but because MPM can be used arthroscopically, both T2-mapping and MPM can be performed in vivo, offering nondestructive means to assess collagen orientation that could be used to obtain longitudinal data in cartilage repair studies.

Preclinical Toxicology Studies of Recombinant Human Platelet-Derived Growth Factor-BB Either Alone or in Combination with Beta-Tricalcium Phosphate and Type I Collagen.

Human platelet-derived growth factor-BB (hPDGF-BB) is a basic polypeptide growth factor released from platelets at the injury site. It is a multifunctional molecule that regulates DNA synthesis and cell division and induces biological effects that are implicated in tissue repair, atherosclerosis, inflammatory responses, and neoplastic diseases. This paper is an overview of the toxicology data generated from a broad testing platform to determine bone, soft tissue, and systemic responses following administration of rhPDGF-BB. Moreover, the systemic and local toxicity of recombinant human PDGF-BB (rhPDGF-BB) in combination with either beta-tricalcium phosphate (ß-TCP) or collagen combined with ß-TCP was studied to determine dermal sensitization, irritation, intramuscular tissue responses, pyrogenicity, genotoxicity, and hemolytic properties. All data strongly suggest that rhPDGF-BB either alone or in combination with ß-TCP or collagen with ß-TCP is biocompatible and has neither systemic nor local toxicity, supporting its safe use in enhancing wound healing in patients.

Teriparatide therapy and beta-tricalcium phosphate enhance scaffold reconstruction of mouse femoral defects.

To investigate the efficacy of endocrine parathyroid hormone treatment on tissue-engineered bone regeneration, massive femoral defects in C57Bl/6 mice were reconstructed with either 100:0 or 85:15 poly-lactic acid (PLA)/beta-tricalcium phosphate (ß-TCP) scaffolds (hereafter PLA or PLA/ßTCP, respectively), which were fabricated with low porosity (<30%) to improve their structural rigidity. Experimental mice were treated starting at 1 week postop with daily subcutaneous injections of 40 µg/kg teriparatide until sacrifice at 9 weeks, whereas control mice underwent the same procedure but were injected with sterile saline. Bone regeneration was assessed longitudinally using planar X-ray and quantitative microcomputed tomography, and the reconstructed femurs were evaluated at 9 weeks either histologically or biomechanically to determine their torsional strength and rigidity. Teriparatide treatment increased bone volume and bone mineral content significantly at 6 weeks and led to enhanced trabeculated bone callus formation that appeared to surround and integrate with the scaffold, thereby establishing union by bridging bone regeneration across the segmental defect in 30% of the reconstructed femurs, regardless of the scaffold type. However, the bone volume and mineral content in the PLA reconstructed femurs treated with teriparatide was reduced at 9 weeks to control levels, but remained significantly increased in the PLA/ßTCP scaffolds. Further, bridged teriparatide-treated femurs demonstrated a prototypical brittle bone torsion behavior, and were significantly stronger and stiffer than control specimens or treated specimens that failed to form bridging bone union. Taken together, these observations suggest that intermittent, systemic parathyroid hormone treatment can enhance bone regeneration in scaffold-reconstructed femoral defects, which can be further enhanced by mineralized (ßTCP) particles within the scaffold.

Evaluation of dense polylactic acid/beta-tricalcium phosphate scaffolds for bone tissue engineering.

Advances in biomaterial fabrication have introduced numerous innovations in designing scaffolds for bone tissue engineering. Often, the focus has been on fabricating scaffolds with high and interconnected porosity that would allow for cellular seeding and tissue ingrowth. However, such scaffolds typically lack the mechanical strength to sustain in vivo ambulatory stresses in models of load bearing cortical bone reconstruction. In this study, we investigated the microstructural and mechanical properties of dense PLA and PLA/beta-TCP (85:15) scaffolds fabricated using a rapid volume expansion phase separation technique, which embeds uncoated beta-TCP particles within the porous polymer. PLA scaffolds had a volumetric porosity in the range of 30 to 40%. With the embedding of beta-TCP mineral particles, the porosity of the scaffolds was reduced in half, whereas the ultimate compressive and torsional strength were significantly increased. We also investigated the properties of the scaffolds as delivery vehicles for growth factors in vitro and in vivo. The low-surface porosity resulted in sub optimal retention efficiency of the growth factors, and burst release kinetics reflecting surface coating rather than volumetric entrapment, regardless of the scaffold used. When loaded with BMP2 and VEGF and implanted in the quadriceps muscle, PLA/beta-TCP scaffolds did not induce ectopic mineralization but induced a significant 1.8-fold increase in neo vessel formation. In conclusion, dense PLA/beta-TCP scaffolds can be engineered with enhanced mechanical properties and potentially be exploited for localized therapeutic factor delivery.

Recombinant human platelet-derived growth factor BB (rhPDGF-BB) and beta-tricalcium phosphate/collagen matrix enhance fracture healing in a diabetic rat model.

Diabetes mellitus is a common systemic disease that has been associated with poor fracture healing outcomes. The mechanism through which diabetes impairs bone regeneration is unknown. One possible mechanism may be related to either decreased or uncoordinated release of local growth factors at the fracture site. Indeed, previous studies have found reduced platelet-derived growth factor (PDGF) levels in the fracture callus of diabetic rats, suggesting that local application of PDGF may overcome the negative effects of diabetes and promote fracture healing. To test this hypothesis, low (22 microg) and high (75 ug) doses of recombinant human PDGF-BB (rhPDGF-BB) were applied directly to femur fracture sites in BB Wistar diabetic rats that were then compared to untreated or vehicle-treated animals. rhPDGF-BB treatment significantly increased early callus cell proliferation compared to that in control specimens. Low dose rhPDGF-BB treatment significantly increased callus peak torque values (p < 0.05) at 8 weeks after fracture as compared to controls. High dose rhPDGF-BB treatment increased callus bone area at 12 weeks postfracture. These data indicate that rhPDGF-BB treatment ameliorates the effects of diabetes on fracture healing by promoting early cellular proliferation that ultimately leads to more bone formation. Local application of rhPDGF-BB may be a new therapeutic approach to treat diabetes-impaired fracture healing.

Combined effects of scaffold stiffening and mechanical preconditioning cycles on construct biomechanics, gene expression, and tendon repair biomechanics.

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC-collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits of stimulation among repairs postsurgery. Our findings emphasize the importance of properly preconditioning a scaffold to better control/modulate MSC differentiation in vitro and to further enhance repair outcome in vivo.

Tensile stimulation of murine stem cell-collagen sponge constructs increases collagen type I gene expression and linear stiffness.

The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 x 10(6) mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h=day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary the mechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.

Accelerated fracture healing in the geriatric, osteoporotic rat with recombinant human platelet-derived growth factor-BB and an injectable beta-tricalcium phosphate/collagen matrix.

Aging and osteoporosis contribute to decreased bone mass and bone mineral density as well as compromised fracture healing rates and bone repair quality. Consequently, the purpose of this study was to determine if recombinant human platelet-derived growth factor-BB (rhPDGF-BB) delivered in an injectable beta-tricalcium phosphate/collagen matrix would enhance tibial fracture healing in geriatric (>2 years of age), osteoporotic rats. A total of 80 rats were divided equally among four groups: Fracture alone; Fracture plus matrix; Fracture plus matrix and either 0.3 mg/mL or 1.0 mg/mL rhPDGF-BB. At 3 and 5 weeks, rats were euthanized and treatment outcome was assessed histologically, radiographically, biomechanically, and by micro-CT. Results indicated rhPDGF-BB-treated fractures in osteoporotic, geriatric rats caused a statistically significant time-dependent increase in torsional strength 5 weeks after treatment. The healed fractures were equivalent in torsional strength to the contralateral, unoperated tibiae. Data from the study are the first, to our knowledge, to underscore rhPDGF-BB efficacy in an injectable beta-tricalcium phosphate/collagen matrix accelerated fracture repair in a geriatric, osteoporotic rat model.

Improving linear stiffness of the cell-seeded collagen sponge constructs by varying the components of the mechanical stimulus.

In vitro mechanical stimulation has been reported to induce cell alignment and increase cellular proliferation and collagen synthesis. Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases construct stiffness and repair biomechanics after surgery. However, these studies used a single mechanical stimulation profile, the latter composed of multiple components whose individual and combined effects on construct properties remain unknown. Thus, the purpose of this study was to understand the relative importance of a subset of these components on construct stiffness. To try to optimize the resulting mechanical stimulus, we used an iterative process to vary peak strain, cycle number, and cycle repetition while controlling cycle frequency (1 Hz), rise and fall times (25% and 17% of the period, respectively), hours of stimulation/day (8 h/day), and total time of stimulation (12 days). Two levels of peak strain (1.2 % and 2.4%), cycle number (100 and 3000 cycles/day), and cycle repetition (1 and 20) were first examined. Higher levels of peak strain and cycle number were then examined to optimize the stimulus using response surface methodology. Our results indicate that constructs stimulated with 2.4% strain, 3000 cycles/day, and one cycle repetition produced the stiffest constructs. Given the significant positive correlations we have previously found between construct stiffness and repair biomechanics at 12 weeks post-surgery, these in vitro enhancements offer the prospect of further improving repair biomechanics.

Mechanical stimulation of tissue engineered tendon constructs: effect of scaffold materials.

Our group has shown that numerous factors can influence how tissue engineered tendon constructs respond to in vitro mechanical stimulation. Although one study showed that stimulating mesenchymal stem cell (MSC)-collagen sponge constructs significantly increased construct linear stiffness and repair biomechanics, a second study showed no such effect when a collagen gel replaced the sponge. While these results suggest that scaffold material impacts the response of MSCs to mechanical stimulation, a well-designed intra-animal study was needed to directly compare the effects of type-I collagen gel versus type-I collagen sponge in regulating MSC response to a mechanical stimulus. Eight constructs from each cell line (n=8 cell lines) were created in specially designed silicone dishes. Four constructs were created by seeding MSCs on a type-I bovine collagen sponge, and the other four were formed by seeding MSCs in a purified bovine collagen gel. In each dish, two cell-sponge and two cell-gel constructs from each line were then mechanically stimulated once every 5 min to a peak strain of 2.4%, for 8 h/day for 2 weeks. The other dish remained in an incubator without stimulation for 2 weeks. After 14 days, all constructs were failed to determine mechanical properties. Mechanical stimulation significantly improved the linear stiffness (0.048+/-0.009 versus 0.015+/-0.004; mean+/-SEM (standard error of the mean ) N/mm) and linear modulus (0.016+/-0.004 versus 0.005+/-0.001; mean+/-SEM MPa) of cell-sponge constructs. However, the same stimulus produced no such improvement in cell-gel construct properties. These results confirm that collagen sponge rather than collagen gel facilitates how cells respond to a mechanical stimulus and may be the scaffold of choice in mechanical stimulation studies to produce functional tissue engineered structures.