Kirsten R. C. Kinneberg

Chondroitin-6-Sulfate Incorporation and Mechanical Stimulation Increase MSC-Collagen Sponge Construct Stiffness

Using functional tissue engineering principles, our laboratory has produced tendon repair tissue which matches the normal patellar tendon force‐displacement curve up to 32% of failure. This repair tissue will need to withstand more strenuous activities, which can reach or even exceed 40% of failure force. To improve the linear stiffness of our tissue engineered constructs (TECs) and tissue engineered repairs, our lab is incorporating the glycosaminoglycan chondroitin‐6‐sulfate (C6S) into a type I collagen scaffold. In this study, we examined the effect of C6S incorporation and mechanical stimulation cycle number on linear stiffness and mRNA expression (collagen types I and III, decorin and fibronectin) for mesenchymal stem cell (MSC)‐collagen sponge TECs. The TECs were fabricated by inoculating MSCs at a density of 0.14 × 106 cells/construct onto pre‐cut scaffolds. Primarily type I collagen scaffold materials, with or without C6S, were cultured using mechanical stimulation with three different cycle numbers (0, 100, or 3,000 cycles/day). After 2 weeks in culture, TECs were evaluated for linear stiffness and mRNA expression. C6S incorporation and cycle number each played an important role in gene expression, but only the interaction of C6S incorporation and cycle number produced a benefit for TEC linear stiffness.

Effect of Implanting a Soft Tissue Autograft in a Central-Third Patellar Tendon Defect: Biomechanical and Histological Comparisons

Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon autograft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n = 10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After 12 weeks of recovery, the animals were euthanized and their limbs were dedicated to biomechanical (n = 7) or histological (n = 3) evaluations. Only stiffness was improved by treatment with patellar tendon autograft relative to natural healing (p = 0.009). Additionally, neither the patellar tendon autograft nor natural healing repairs regenerated a normal zonal insertion site between the tendon and bone. Immunohistochemical staining for collagen type II demonstrated that fibrocartilage-like tissue was regenerated at the tendon-bone interface for both repairs. However, the tissue was disorganized. Insufficient tissue integration at the tendon-to-bone junction led to repair tissue failure at the insertion site during testing. It is important to re-establish the tendon-to-bone insertion site because it provides joint stability and enables force transmission from muscle to tendon and subsequent loading of the tendon. Without loading, tendon mechanical properties deteriorate. Future studies by our laboratory will investigate potential strategies to improve patellar tendon autograft integration into bone using this model.

The native cell population does not contribute to central-third graft healing at 6, 12, or 26 weeks in the rabbit patellar tendon

Investigators do not yet understand the role of intrinsic tendon cells in healing at the tendon‐to‐bone enthesis. Therefore, our first objective was to understand how the native cell population influences tendon autograft incorporation in the central‐third patellar tendon (PT) defect site. To do this, we contrasted the histochemical and biomechanical properties of de‐cellularized patellar tendon autograft (dcPTA) and patellar tendon autograft (PTA) repairs in the skeletally mature New Zealand white rabbit. Recognizing that soft tissues in many animal models require up to 26 weeks to incorporate into bone, our second objective was to investigate how recovery time affects enthesis formation and graft tissue biomechanical properties. Thus, we examined graft structure and mechanics at 6, 12, and 26 weeks post‐surgery. Our results showed that maintaining the native cell population produced no histochemical or biomechanical benefit at 6, 12, or 26 weeks. These findings suggest that PTA healing is mediated more by extrinsic rather than intrinsic cellular mechanisms. Moreover, while repair tissue biomechanical properties generally increased from 6 to 12 weeks after surgery, no further improvements were noted up to 26 weeks.

Evolving Strategies in Mechanobiology to More Effectively Treat Damaged Musculoskeletal Tissues

In this paper, we had four primary objectives. (1) We reviewed a brief history of the Lissner award and the individual for whom it is named, H.R. Lissner. We examined the type (musculoskeletal, cardiovascular, and other) and scale (organism to molecular) of research performed by prior Lissner awardees using a hierarchical paradigm adopted at the 2007 Biomechanics Summit of the US National Committee on Biomechanics. (2) We compared the research conducted by the Lissner award winners working in the musculoskeletal (MS) field with the evolution of our MS research and showed similar trends in scale over the past 35 years. (3) We discussed our evolving mechanobiology strategies for treating musculoskeletal injuries by accounting for clinical, biomechanical, and biological considerations. These strategies included studies to determine the function of the anterior cruciate ligament and its graft replacements as well as novel methods to enhance soft tissue healing using tissue engineering, functional tissue engineering, and, more recently, fundamental tissue engineering approaches. (4) We concluded with thoughts about future directions, suggesting grand challenges still facing bioengineers as well as the immense opportunities for young investigators working in musculoskeletal research. Hopefully, these retrospective and prospective analyses will be useful as the ASME Bioengineering Division charts future research directions.

MPC-Collagen Gel Biologic Augmentations do not Promote Patellar Tendon Integration Into Bone

The high failure rate of rotator cuff repair is often attributed to the fact that a normal zonal insertion site is not regenerated between tendon and bone. [1,2] Interestingly, a previous study in our laboratory demonstrated that a patellar tendon autograft (PTA) used to repair a central-third patellar tendon (PT) defect also does not regenerate a normal zonal insertion site at 12 weeks following surgery. Therefore, using our model of tendon healing, our objective was to design a biologic augmentation (BA) that could be implemented at the insertion site between tendon and bone to help promote integration. The results of this study have potential application in developing new techniques for rotator cuff repair.Copyright

Regulation of Tendon Tissue Engineered Construct Stiffness by Culture Time, Mesenchymal Stem Cells and Mechanical Stimulation

More than 57.2 million episodes of musculoskeletal injuries were recorded in 2004 in the United States, costing the US economy more than

Evaluation of a Novel Scaffold Material for Tendon Tissue Engineering

254 billion [1, 2]. Approximately 45% of these are tendon, ligament and joint capsule related injuries [3]. Tissue engineering approaches have emerged as an attractive alternative to conventional and oftentimes ineffective treatment methods. Implanting MSC-seeded collagen tissue engineered constructs (TECs) in the central defects of patellar tendon (PT) has significantly improved repair biomechanics compared to natural healing as well as acellular repairs in the rabbit model [4, 5]. Mechanically stimulating these MSC-collagen TECs further improved repair outcome, and TEC stiffness at the time of surgery positively correlated with and predicted repair stiffness twelve weeks after surgery [6, 7]. Although these improvements were observed twelve weeks after surgery, repairs were still not strong enough to withstand forces that might arise during more strenuous activities.Copyright

Combined Effect of Glycosaminoglycan and Mechanical Stimulation on the In Vitro Biomechanics of Tissue Engineered Tendon Constructs

Tissue engineering offers an attractive alternative to direct repair or reconstruction of soft tissue injuries. Tissue engineered constructs containing mesenchymal stem cells (MSCs) seeded in commercially available type I collagen sponges (P1076, Kensey Nash Corporation, Exton, PA) are currently being used within our laboratory to repair tendon injuries in rabbit models [1]. When introduced into the wound site, mechanically stimulated stem cell-collagen sponge constructs exhibit 50% greater maximum force and stiffness at 12 weeks compared to values for static controls [1]. However, these constructs often lack the maximum force sufficient to resist the peak in vivo forces acting on the repair site [2, 3]. Insufficient repair biomechanics can be attributed to the poor initial mechanical resistance provided by the collagen sponges to replace the function of the lost tendon before its degradation and replacement with new extracellular matrix. This current study seeks to identify a biologically-derived scaffold with improved mechanical integrity that could be used in stem cell-based tissue engineered constructs for tendon repair.Copyright

The use of mesenchymal stem cells in collagen-based scaffolds for tissue-engineered repair of tendons

Tissue engineering offers an attractive alternative to direct repair or reconstruction of injuries to tendons, ligaments and capsular structures that represent almost 45% of the 32 million musculoskeletal injuries that occur each year in the United States [1]. Mesenchymal stem cell (MSC)-seeded collagen constructs are currently being used by our group to repair tendon injuries in the rabbit model [2, 3]. Although these cell-assisted repairs exhibit 50% greater maximum force and stiffness at 12 weeks compared to values for natural repair, tissues often lack the maximum force sufficient to resist the peak in vivo forces acting on the repair site [3]. Our laboratory has previously demonstrated that in vitro construct stiffness and repair stiffness at 12 weeks post surgery are positively correlated [4]. Therefore, in an effort to further improve the repair outcome using tissue engineering, we continue our investigation of scaffold materials to create stiffer MSC-collagen constructs. Our group has recently evaluated two scaffold materials, type I collagen sponges fabricated within the Engineered Skin Lab (ESL, Shriners Hospitals for Children) by a freezing and lyophilization process with and without glycosaminoglycan (chondroitin-6-sulfate; GAG) [5] and found the ESL sponges to significantly improve biomechanical properties of the constructs compared to sponges we currently use in the lab (P1076, Kensey Nash Corporation, Exton, PA). This study also demonstrated that GAG significantly upregulates collagen type I, decorin, and fibronectin gene expression (unpublished results).Copyright