Fibrochondrogenic differentiation of mesenchymal stem cells through chemical stimulation

Faculty Development Competitive Research Grant Program 2018-2020

Injuries associated with tendons are among the most common trauma, with over 250,000 rotator cuff tendon repairs performed annually in the United States only1. They often occur at the regions close to the tendon-bone interface, and are susceptible to incomplete healing resulting in compositionally and structurally abnormal tissue2,3. The native tendon-bone interface is comprised of gradients of diverse tissues, namely, tendon, fibrocartilage and bone, containing multiple cell phenotypes such as fibroblasts, fibrochondrocytes and osteoblasts in respective zones4,5. The fibrocartilage tissue is further divided into non-calcified and calcified regions. It was demonstrated that the mechanical fixation of current tendon reconstruction grafts often fail to reestablish this hierarchical soft tissue-to-bone transition post-surgery6-9. Therefore, there is a need for the new augmentation matrices to improve the biological fixation and scar-free healing at the tendon-bone interface.
To this end, our approach to tendon-bone integration focuses on the use of biomimetic, nanofiber-based scaffolds that will enable interface regeneration through controlled release of key bioactive agents. It has been reported that growth factors injected into the zone of injury may facilitate healing and restoration of the normal function of the tendon-bone interface10,11. In fact, transforming growth factor - beta 3 (TGF-β3) is upregulated during the development of the tendon-to-bone insertion12. In order to enhance the tendon-to-bone healing process, the objective of this proposal is to 1) establish controlled release of bioactive TGF-β3 from the nanofiber scaffolds, and 2) to optimize growth factor release kinetics by controlling fiber diameter relying upon the effect of released TGF-β3 on the potential of human mesenchymal stem cells (MSCs) to regenerate a tendon-to-bone like interface. Our working hypothesis is that TGF-β3 incorporated into nanofibers will remain stable and bioactive during processing and be released in a sustained manner, the dosage being controlled by fiber diameter. Furthermore, TGF-β3 released from scaffolds will be more effective on enhancing the biological activity of MSCs as compared with exogenous control, and its efficacy will be optimized by fiber diameter based on its performance for interface-related matrix formation (collagen and glycosaminoglycans) and expression of markers such as collagen types I, II, and X by MSCs.
Specifically, the hypotheses and related aims of this proposal are:
Hypothesis 1:The TGF-β3 will release from nanofibers in a sustained manner and will remain stable and bioactive.
Aim 1a:Determine the release kinetics of TGF-β3 from poly(lactic-co-glycolic acid), PLGA, nanofibers and its stability over time.
Aim 1b:Determine the bioactivity of TGF-β3 on the in vitro response of MSCs.
Hypothesis 2:The TGF-β3 release and bioactivity of MSCs will be controlled by scaffold fiber diameter.
Aim 2a:Determine the effect of nanofiber diameter on release kinetics of TGF-β3.
Aim 2b:Optimize the diameter-dependent effectiveness of TGF-β3 on MSC activity.

This proposal aims at determining 1) the release kinetics and stability of TGF-β3 (Aim 1a), as well as its bioactivity on the response of MSCs (Aim 1b), and 2) the diameter-dependent release (Aim 2a) and efficacy of TGF-β3 on MSC-mediated regeneration of the tendon-to-bone interface (Aim 2b). It is anticipated that findings of our planned studies will not only reveal the importance of design and use of nanotechnology-driven biomimetic scaffolds in tissue engineering but also yield new insights into the effect of bioactive molecules on interface regeneration when the local bioavailability of these factors are controlled. These discoveries will serve as the foundation for the development of biomimetic tissue engineering technologies aimed at promoting biological graft fixation and integrative soft tissue repair that remains as a significant societal challenge for meeting the need of over 250,000 patients suffering from rotator cuff tendon related injuries.

SIGNIFICANCE
Rotator cuff tears are among the most common injuries afflicting the shoulder, and clinical intervention is required because rotator cuff injuries do not heal themselves, largely due to the complex anatomy and structure (Fig. 2.1), extended range of motion of the shoulder joint, as well as the relative weakening and hypovascularization of the cuff tendons13-15. Early primary anatomic repair followed by carefully controlled rehabilitation is currently the treatment of choice for symptomatic rotator cuff tears15. Cuff repair has evolved from traditional open repair to "mini-open" to primarily arthroscopic16-20, and these methods have significantly improved biomechanical strength and graft stability21. Attention has now turned to addressing the biological challenge of achieving cuff healing to the anatomic bony footprint, which is essential for tendon-bone integration and long term clinical success.
Primary repair of chronic degenerative cuff injuries often result in excessive tension on the cuff tissues and at the repair site15,22,23. Failure rates as high as 90% have been reported after primary repair of chronic rotator cuff injuries17. To improve healing, synthetic tendon grafts24,25 were designed to reconstruct large rotator cuff defects, but with limited success. Biological matrices such as acellularized allogeneic and xenogeneic extracellular matrix scaffolds have recently emerged as promising grafts for rotator cuff repair15,26. Furthermore, graft patches used in rotator cuff repair are primarily utilized for enhancing the biologic process of tendon healing, and highly promising results were reported for small intestinal submucosa (SIS) in animal models15,26. SIS is particularly attractive as it exhibits biomimetic, collagen nanofiber-based architecture and alignment27. Unfortunately suboptimal outcome were observed in human trials28,29. Recently, Iannotti et al. performed a randomized controlled trial and observed that augmentation with SIS did not improve the rate of tendon healing or clinical outcome scores. Similar findings have been reported for other biological grafts used in cuff repair28,30. The suboptimal results of biologically-derived grafts may be attributed to mismatch in mechanical properties and the rapid matrix remodeling experienced in the physiologically demanding and often diseased shoulder joint. Therefore, the debilitating effect of rotator cuff tears coupled with the high incidence of failure associated with existing graft choices emphasize the clinical need for functional rotator graft augmentation solutions.
While the mechanism for interface regeneration is not known, knowledge of the development process provides valuable cues for its regeneration. In this regard, TGF-β3 was shown to act as signaling molecules during embryonic tendon/ligament formation and has been reported to be upregulated during the formation of the tendon-bone insertion site11,12. In addition, growth factors promoted the biologic repair and augmentation of the tendon-to-bone healing at the rotator cuff11,31,32. These observations collectively suggest that presence of TGF-β3 is a critical design parameter to be considered for interface tissue engineering.

INNOVATION
The emphasis in the field of tissue engineering has shifted from tissue formation to function33, and a major challenge in this effort is how to achieve biological fixation of engineered grafts to tendon and bone repair sites. Nanofiber-based scaffolds represent promising matrices for interface tissue engineering due to their superior biomimetic potential and physiological relevance, as they closely mimic the extracellular matrix34-38. Moreover, by controlling the spatial and temporal bioavailability of key growth factors through the nanofibers, we can accelerate or promote functional regeneration of the tendon-to-bone interface.
Controlled release of biomolecules makes them available to cells in a sustained manner, which can more efficiently regulate their behavior. As noted earlier, TGF-β3 is upregulated during the development stage of tendon-to-bone interface12, and incorporating it into nanofibers and releasing during culture may induce synthesis of interface related extracellular matrix components. To this end, we have earlier developed aligned nanofiber-based scaffold incorporated with TGF-β3, and it is anticipated that sustained TGF-β3 release will promote interface regeneration.
Our goal is to utilize the nanofiber delivery system to induce mesenchymal stem cell-mediated interface regeneration. The hMSCs have the capacity to differentiate into multiple lineages once treated with appropriate chemical stimulations. It is well established that bone marrow derived MSCs can be manipulated to express and produce collagen types I, II, and X when chemically stimulated with TGF-β3.39,40 Based on these findings, we expect that the fibrous scaffolds incorporated with TGF-β3 will regulate the behavior of MSCs to express interface related markers such as collagen types I, II, and X (Fig. 2.1) and produce relevant matrix components.
Controlling and optimizing the release of TGF-β3 as well as its efficacy on biological activity of MSCs by using fiber diameter as an independent variable is another component of our innovative scaffold design. Based on the theory of diffusion41, which predicts fiber diameter dependency of release, we propose that release rate of TGF-β3 from these scaffold systems as well as biological activity of MSCs cultured on these scaffolds will be controlled by fiber diameter.
MSCs represent pluripotent and autologous source of cells for tissue engineering.42,43 Furthermore, MSCs have been investigated as a potential cell source for bone44, cartilage45, and tendon43 tissue engineering. With the innovative delivery system of this investigation, we are aiming to guide MSCs to synthesize fibrocartilaginous matrix components. Therefore, the completion of our work should demonstrate the efficacy of delivery system for MSCs to selectively develop into chondrogenic and fibroblastic phenotypes concomitantly.