Fabrication of Chimeric Hair Follicles for Skin Tissue Engineering.

Fabrication of engineered skin substitutes provides an alternative approach for the treatment of full-thickness burns and other skin injuries. Improving the functionality of current skin substitute models requires incorporation of skin appendages, including hair follicles, sebaceous glands, and sweat glands. In this chapter, methods for generating skin substitutes incorporating chimeric hair follicles are described. Isolation of human keratinocytes, human fibroblasts, and murine dermal papilla cells is first outlined. These cell types are then combined with collagen-glycosaminoglycan (GAG) scaffolds to generate human-murine chimeric grafts which are then grafted to full-thickness surgical wounds in immunodeficient mice. The methods described allow for the generation of a human-mouse follicular structure.

Tissue engineering of skin and regenerative medicine for wound care.

Engineering of biologic skin substitutes has progressed over time from individual applications of skin cells, or biopolymer scaffolds, to combinations of cells and scaffolds for treatment, healing, and closure of acute and chronic skin wounds. Skin substitutes may be categorized into three groups: acellular scaffolds, temporary substitutes containing allogeneic skin cells, and permanent substitutes containing autologous skin cells. Combined use of acellular dermal substitutes with permanent skin substitutes containing autologous cells has been shown to provide definitive wound closure in burns involving greater than 90% of the total body surface area. These advances have contributed to reduced morbidity and mortality from both acute and chronic wounds but, to date, have failed to replace all of the structures and functions of the skin. Among the remaining deficiencies in cellular or biologic skin substitutes are hypopigmentation, absence of stable vascular and lymphatic networks, absence of hair follicles, sebaceous and sweat glands, and incomplete innervation. Correction of these deficiencies depends on regulation of biologic pathways of embryonic and fetal development to restore the full anatomy and physiology of uninjured skin. Elucidation and integration of developmental biology into future models of biologic skin substitutes promises to restore complete anatomy and physiology, and further reduce morbidity from skin wounds and scar. This article offers a review of recent advances in skin cell thrapies and discusses the future prospects in cutaneous regeneration.

The LG/J murine strain exhibits near-normal tendon biomechanical properties following a full-length central patellar tendon defect.

PURPOSE OF THE STUDY: Identifying biological success criteria is needed to improve therapies, and one strategy for identifying them is to analyze the RNA transcriptome for successful and unsuccessful models of tendon healing. We have characterized the MRL/MpJ murine strain and found improved mechanical outcomes following a central patellar tendon (PT) injury. In this study, we evaluate the healing of the LG/J murine strain, which comprises 75% of the MRL/MpJ background, to determine if the LG/J also exhibits improved biomechanical properties following injury and to determine differentially expressed transcription factors across the C57BL/6, MRL/MpJ and the LG/J strains during the early stages of healing. MATERIALS AND METHODS: A full-length, central PT defect was created in 16-20 week old MRL/MpJ, LG/J, and C57BL/6 murine strains. Mechanical properties were assessed at 2, 5, and 8 weeks post surgery. Transcriptomic expression was assessed at 3, 7, and 14 days following injury using a novel clustering software program to evaluate differential expression of transcription factors. RESULTS: Average LG/J structural properties improved to 96.7% and 97.2% of native LG/J PT stiffness and ultimate load by 8 weeks post surgery, respectively. We found the LG/J responded by increasing expression of transcription factors implicated in the inflammatory response and collagen fibril organization. CONCLUSIONS: The LG/J strain returns to normal structural properties by 8 weeks, with steadily increasing properties at each time point. Future work will characterize the cell populations responding to injury and investigate the role of the differentially expressed transcription factors during healing.

Improved biomechanical and biological outcomes in the MRL/MpJ murine strain following a full-length patellar tendon injury.

Musculoskeletal injuries greatly affect the U.S. population and current clinical approaches fail to restore long-term native tissue structure and function. Tissue engineering is a strategy advocated to improve tendon healing; however, the field still needs to establish biological benchmarks for assessing the effectiveness of tissue-engineered structures. Investigating superior healing models, such as the MRL/MpJ, offers the opportunity to first characterize successful healing and then apply experimental findings to tissue-engineered therapies. This study seeks to evaluate the MRL/MpJ's healing response following a central patellar tendon injury compared to wildtype. Gene expression and histology were assessed at 3, 7, and 14 days following injury and mechanical properties were measured at 2, 5, and 8 weeks. Native patellar tendon biological and mechanical properties were not different between strains. Following injury, the MRL/MpJ displayed increased mechanical properties between 5 and 8 weeks; however, early tenogenic expression patterns were not different between the strains. Furthermore, expression of the cyclin-dependent kinase inhibitor, p21, was not different between strains, suggesting an alternative mechanism may be driving the healing response. Future studies will investigate collagen structure and alignment of the repair tissue and characterize the complete healing transcriptome to identify mechanisms driving the MRL/MpJ response.

Functional tissue engineering of tendon: Establishing biological success criteria for improving tendon repair.

Improving tendon repair using Functional Tissue Engineering (FTE) principles has been the focus of our laboratory over the last decade. Although our primary goals were initially focused only on mechanical outcomes, we are now carefully assessing the biological properties of our tissue-engineered tendon repairs so as to link biological influences with mechanics. However, given the complexities of tendon development and healing, it remains challenging to determine which aspects of tendon biology are the most important to focus on in the context of tissue engineering. To address this problem, we have formalized a strategy to identify, prioritize, and evaluate potential biological success criteria for tendon repair. We have defined numerous biological properties of normal tendon relative to cellular phenotype, extracellular matrix and tissue ultra-structure that we would like to reproduce in our tissue-engineered repairs and prioritized these biological criteria by examining their relative importance during both normal development and natural tendon healing. Here, we propose three specific biological criteria which we believe are essential for normal tendon function: (1) scleraxis-expressing cells; (2) well-organized and axially-aligned collagen fibrils having bimodal diameter distribution; and (3) a specialized tendon-to-bone insertion site. Moving forward, these biological success criteria will be used in conjunction with our already established mechanical success criteria to evaluate the effectiveness of our tissue-engineered tendon repairs.

The role of mechanical loading in tendon development, maintenance, injury, and repair.

Tendon injuries often result from excessive or insufficient mechanical loading, impairing the ability of the local tendon cell population to maintain normal tendon function. The resident cell population composing tendon tissue is mechanosensitive, given that the cells are able to alter the extracellular matrix in response to modifications of the local loading environment. Natural tendon healing is insufficient, characterized by improper collagen fibril diameter formation, collagen fibril distribution, and overall fibril misalignment. Current tendon repair rehabilitation protocols focus on implementing early, well-controlled eccentric loading exercises to improve repair outcome. Tissue engineers look toward incorporating mechanical loading regimens to precondition cell populations for the creation of improved biological augmentations for tendon repair.

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.