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1.  The role of collagen crosslinks in ageing and diabetes - the good, the bad, and the ugly 
The non-enzymatic reaction of proteins with glucose (glycation) is a topic of rapidly growing importance in human health and medicine. There is increasing evidence that this reaction plays a central role in ageing and disease of connective tissues. Of particular interest are changes in type-I collagens, long-lived proteins that form the mechanical backbone of connective tissues in nearly every human organ. Despite considerable correlative evidence relating extracellular matrix (ECM) glycation to disease, little is known of how ECM modification by glucose impacts matrix mechanics and damage, cell-matrix interactions, and matrix turnover during aging. More daunting is to understand how these factors interact to cumulatively affect local repair of matrix damage, progression of tissue disease, or systemic health and longevity. This focused review will summarize what is currently known regarding collagen glycation as a potential driver of connective tissue disease. We concentrate attention on tendon as an affected connective tissue with large clinical relevance, and as a tissue that can serve as a useful model tissue for investigation into glycation as a potentially critical player in tissue fibrosis related to ageing and diabetes.
PMCID: PMC4241420  PMID: 25489547
collagen; advanced glycation end-products; crosslinks; tendon mechanics; diabetes; ageing
2.  Advanced Glycation End-Products Reduce Collagen Molecular Sliding to Affect Collagen Fibril Damage Mechanisms but Not Stiffness 
PLoS ONE  2014;9(11):e110948.
Advanced glycation end-products (AGE) contribute to age-related connective tissue damage and functional deficit. The documented association between AGE formation on collagens and the correlated progressive stiffening of tissues has widely been presumed causative, despite the lack of mechanistic understanding. The present study investigates precisely how AGEs affect mechanical function of the collagen fibril – the supramolecular functional load-bearing unit within most tissues. We employed synchrotron small-angle X-ray scattering (SAXS) and carefully controlled mechanical testing after introducing AGEs in explants of rat-tail tendon using the metabolite methylglyoxal (MGO). Mass spectrometry and collagen fluorescence verified substantial formation of AGEs by the treatment. Associated mechanical changes of the tissue (increased stiffness and failure strength, decreased stress relaxation) were consistent with reports from the literature. SAXS analysis revealed clear changes in molecular deformation within MGO treated fibrils. Underlying the associated increase in tissue strength, we infer from the data that MGO modified collagen fibrils supported higher loads to failure by maintaining an intact quarter-staggered conformation to nearly twice the level of fibril strain in controls. This apparent increase in fibril failure resistance was characterized by reduced side-by-side sliding of collagen molecules within fibrils, reflecting lateral molecular interconnectivity by AGEs. Surprisingly, no change in maximum fibril modulus (2.5 GPa) accompanied the changes in fibril failure behavior, strongly contradicting the widespread assumption that tissue stiffening in ageing and diabetes is directly related to AGE increased fibril stiffness. We conclude that AGEs can alter physiologically relevant failure behavior of collagen fibrils, but that tissue level changes in stiffness likely occur at higher levels of tissue architecture.
PMCID: PMC4217736  PMID: 25364829
3.  Prevention of Peritendinous Adhesions Using an Electrospun DegraPol Polymer Tube: A Histological, Ultrasonographic, and Biomechanical Study in Rabbits 
BioMed Research International  2014;2014:656240.
Purpose. One of the great challenges in surgical tendon rupture repair is to minimize peritendinous adhesions. In order to reduce adhesion formation, a physical barrier was applied to a sutured rabbit Achilles tendon, with two different immobilization protocols used postoperatively. Methods. Thirty New Zealand white rabbits received a laceration on the Achilles tendon, sutured with a 4-strand Becker suture, and half of the rabbits got a DegraPol tube at the repair site. While fifteen rabbits had their treated hind leg in a 180° stretched position during 6 weeks (adhesion provoking immobilization), the other fifteen rabbits were recasted with a 150° position after 3 weeks (adhesion inhibiting immobilization). Adhesion extent was analysed macroscopically, via ultrasound and histology. Inflammation was determined histologically. Biomechanical properties were analysed. Results. Application of a DegraPol tube reduced adhesion formation by approximately 20%—independently of the immobilization protocol. Biomechanical properties of extracted specimen were not affected by the tube application. There was no serious inflammatory reaction towards the implant material. Conclusions. Implantation of a DegraPol tube tightly set around a sutured tendon acts as a beneficial physical barrier and prevents adhesion formation significantly—without affecting the tendon healing process.
PMCID: PMC4101979  PMID: 25101292
4.  Differences between the Cell Populations from the Peritenon and the Tendon Core with Regard to Their Potential Implication in Tendon Repair 
PLoS ONE  2014;9(3):e92474.
The role of intrinsic and extrinsic healing in injured tendons is still debated. In this study, we characterized cell plasticity, proliferative capacity, and migration characteristics as proxy measures of healing potential in cells derived from the peritenon (extrinsic healing) and compared these to cells from the tendon core (intrinsic healing). Both cell populations were extracted from horse superficial digital flexor tendon and characterized for tenogenic and matrix remodeling markers as well as for rates of migration and replication. Furthermore, colony-forming unit assays, multipotency assays, and real-time quantitative polymerase chain reaction analyses of markers of osteogenic and adipogenic differentiation after culture in induction media were performed. Finally, cellular capacity for differentiation towards a myofibroblastic phenotype was assessed. Our results demonstrate that both tendon- and peritenon-derived cell populations are capable of adipogenic and osteogenic differentiation, with higher expression of progenitor cell markers in peritenon cells. Cells from the peritenon also migrated faster, replicate more quickly, and show higher differentiation potential toward a myofibroblastic phenotype when compared to cells from the tendon core. Based on these data, we suggest that cells from the peritenon have substantial potential to influence tendon-healing outcome, warranting further scrutiny of their role.
PMCID: PMC3961373  PMID: 24651449
5.  Smad8/BMP2–Engineered Mesenchymal Stem Cells Induce Accelerated Recovery of the Biomechanical Properties of the Achilles Tendon 
Tendon tissue regeneration is an important goal for orthopedic medicine. We hypothesized that implantation of Smad8/BMP2–engineered MSCs in a full-thickness defect of the Achilles tendon (AT) would induce regeneration of tissue with improved biomechanical properties. A 2 mm defect was created in the distal region of murine ATs. The injured tendons were then sutured together or given implants of genetically engineered MSCs (GE group), nonengineered MSCs (CH3 group), or fibrin gel containing no cells (FG group). Three weeks later the mice were killed, and their healing tendons were excised and processed for histological or biomechanical analysis. A biomechanical analysis showed that tendons that received implants of genetically engineered MSCs had the highest effective stiffness (> 70% greater than natural healing, p < 0.001) and elastic modulus. There were no significant differences in either ultimate load or maximum stress among the treatment groups. Histological analysis revealed a tendon-like structure with elongated cells mainly in the GE group. ATs that had been implanted with Smad8/BMP2–engineered stem cells displayed a better material distribution and functional recovery than control groups. While additional study is required to determine long-term effects of GE MSCs on tendon healing, we conclude that genetically engineered MSCs may be a promising therapeutic tool for accelerating short-term functional recovery in the treatment of tendon injuries.
PMCID: PMC3479351  PMID: 22696396
Tendon repair; Smad8/BMP2; Tissue engineering; Biomechanics; Achilles tendon
6.  Paracrine Interactions between Mesenchymal Stem Cells Affect Substrate Driven Differentiation toward Tendon and Bone Phenotypes 
PLoS ONE  2012;7(2):e31504.
We investigated substrate dependent paracrine signaling between subpopulations of bone marrow stromal cells (BMSCs) that may affect the formation, or perhaps malformation, of the regenerating tendon to bone enthesis. Polyacrylamide substrates approximating the elastic modulus of tendon granulation tissue and the osteoid of healing bone (10–90 kPa) were functionalized with whole length fibronectin (Fn), type-I collagen (Col), or a mixed ligand solution (Fn/Col), and BMSCs were cultured in growth media alone or media supplemented with soluble Col or Fn. More rigid substrates with a narrow mechanical gradient (70–90 kPa) robustly induced osteogenic cell differentiation when functionalized with either Col or Fn. On broader mechanical gradient substrates (with a linear elastic modulus gradient from 10–90 kPa), cell differentiation was markedly osteogenic on subregions of Fn functionalized substrates above 20 kPa, but osteogenic activity was inhibited on all subregions of Col substrates. Osteogenic behavior was not observed when cells were cultured on Fn substrates if Col was present either in the media or on the substrate (Fn/Col). Tenogenic differentiation markers were observed only on Col substrates with moderate rigidity (∼30–50 kPa). Tenogenic differentiation was unaltered by soluble or substrate bound Fn. Co-culture of narrow gradient subsections revealed that any inclusion of tenogenic substrates (30–50 kPa, Col), caused otherwise osteogenic substrates to not develop markers of osteogenic differentiation, while increasing cell proliferation. These apparently paracrine effects could be mediated by bone morphogenetic protein-2 (BMP-2), as first confirmed by gene-level expression of BMP-2 and the transcription factor Smad8, and verified by BMP-2 media supplementation at levels similar to observed cell-secreted concentrations, which arrested osteogenic differentiation in 14 day cultures. Thus, cell instructive biomaterials with engineered mechanical and biochemical properties represent potentially powerful tools for directing BMSC differentiation to tendon and bone, however paracrine signals from tenogenic cells may delay osteogenesis at the healing enthesis.
PMCID: PMC3280320  PMID: 22355373
7.  Genetically Modified Mesenchymal Stem Cells Induce Mechanically Stable Posterior Spine Fusion 
Tissue Engineering. Part A  2010;16(12):3679-3686.
Most spine fusion procedures involve the use of prosthetic fixation devices combined with autologous bone grafts rather than biological treatment. We had shown that spine fusion could be achieved by injection of bone morphogenetic protein-2 (BMP-2)-expressing mesenchymal stem cells (MSCs) into the paraspinal muscle. In this study, we hypothesized that posterior spinal fusion achieved using genetically modified MSCs would be mechanically comparable to that realized using a mechanical fixation. BMP-2-expressing MSCs were injected bilaterally into paravertebral muscles of the mouse lumbar spine. In one control group BMP-2 expression was inhibited. Microcomputed tomography and histological analyses were used to evaluate bone formation. For comparison, a group of mouse spines were bilaterally fused with stainless steel pins. The harvested spines were later tested using a custom four-point bending apparatus and structural bending stiffness was estimated. To assess the degree to which MSC vertebral fusion was targeted and to quantify the effects of fusion on adjacent spinal segments, images of the loaded spine curvature were analyzed to extract rigidity of the individual spinal segments. Bone bridging of the targeted vertebrae was observed in the BMP-2-expressing MSC group, whereas no bone formation was noted in any control group. The biomechanical tests showed that MSC-mediated spinal fusion was as effective as stainless steel pin-based fusion and significantly more rigid than the control groups. Local analysis showed that the distribution of stiffness in the MSC-based fusion group was similar to that in the steel pin fusion group, with the majority of spinal stiffness contributed by the targeted fusion at L3–L5. Our findings demonstrate that MSC-induced spinal fusion can convey biomechanical rigidity to a targeted segment that is comparable to that achieved using an instrumental fixation.
PMCID: PMC2991214  PMID: 20618082

Results 1-7 (7)