Assistant Professor Oregon Health & Science University Portland, Oregon, United States
Introduction:: Limb threatening injuries, with composite bone and soft tissue loss, require complex, staged surgical management and often corrective surgeries and physical therapy. Open fractures with soft tissue damage are 4-5 times more likely to result in delayed or failed bone union. Moreover, the tissue that is transferred as a replacement for lost muscle, acts mostly as a volume filler and often does not restore muscular strength or loading ability. Unfortunately, patients often have poor long-term limb function, severe disability, impaired gait, chronic pain, and frequent delayed amputation. The current best surgical techniques do not functionally heal muscle to fully restore limb function. However, the advancement of tissue engineering strategies and exercise rehabilitation has the potential to guide and unlock a new pathway of restorative care for these patients. Our group utilizes nanoscale patterned scaffolds with tunable biophysical properties to guide the structure-function relationship within muscle to facilitate healing. Furthermore, we have designed materials that synergistically work with exercise stimulation to improve recovery and repair following severe musculoskeletal trauma. These collective studies will describe the integration of biophysical cues on the subcellular level using nanomaterials and on the whole system level using exercise to achieve additive regenerative benefits.
Materials and Methods:: Scaffold Fabrication: Extracellular matrix (ECM)-based scaffolds were fabricated by shear extrusion, a process that organizes the collagen nanofibrils in the direction of extrusion. Briefly, Collagen Type I or ECM was dialyzed to 35 mg/ml and extruded (22G needle) into a warmed (37C) 10X PBS solution then stretched across hydrophobic glass and dried overnight.
Cell Culture: Vitro studies included primary GFP+ mouse myoblasts, C2C12 myoblasts, MC3T3 pre-osteoblasts, and primary human microvascular endothelial cells. Cells were seeded at 500k cells per scaffold and evaluated for myogenesis (myotube length, percentage), osteogenesis (Alkaline phosphatase activity, alizarin red staining), and endothelial phenotype (CD31/VCAM-1 expression, junction integrity).
Murine injury models: A volumetric muscle loss (VML) model was created by surgical removal of 20-30% of the tibilais anterior muscle. A composite injury model was generated by creating a 2 mm segmental tibia defect (using a dental drill) adjacent to a VML injury. Engineered muscle scaffolds were transplanted into the muscle defect site and recovery was assessed between 21-56 days post transplant.
Exercise regimen: Animals were acclimated to free running wheels (Lafayette) for 72 hours prior to surgery and baseline running behavior was collected (average distance/day) for normalization across groups. Following surgery, mice were returned to static housing to recover and re-introduced to running wheels at day 7 post-surgery until day 21 or 28.
Healing assessments: Bone healing (microCT/mRUST, bone mineral density, histology); Muscle healing muscle force production, histology); Overall health status and limb recovery (survival rate, limb morbidity score, dynamic weight bearing, CBCs, circulating cytokine levels).
Results, Conclusions, and Discussions:: In order to strategize a high-level regenerative approach to mediate healing, we sought to engineer the biophysical and biomechanical musculoskeletal response.
We established a method to control the biophysical properties of extruded collagen and ECM-based fibrillar scaffolds (anisotropy, fiber diameter, porosity and stiffness). These properties differentially directed the cytoskeletal organization and phenotypic determination of myogenic, osteogenic and endothelial cells. For example, myotube length and fusion index increased as fiber diameters and porosity decreased. In vitro, anisotropic scaffolds improved the differentiation efficiency of skeletal muscle myoblasts (2-fold increase in myotube length, 3-fold increase in nuclei/myotube, 20% more mature myotubes), and displayed greater myotube force properties (4-fold increased max contraction velocity, 90% greater contraction area) compared to scaffolds with decreased anisotropy and stiffness.
In vivo, engineered muscle scaffolds improved tissue regeneration in a mouse model of volumetric muscle loss (VML) as well as improved functional muscle recovery and limb healing in a bone-muscle composite injury. Transplanted nano-patterned engineered muscle scaffolds, promoted extensive de novo myogenesis and angiogenesis, increased muscle mass and recapitulated the structural organization of native parallel-aligned myofibers in the VML injury. In complex composite bone-muscle injuries, these scaffolds better restored the TA muscle cross-sectional area (p < 0.01) and a maximum contractile force (p < 0.05), increased survival rate (from 68% to 83%) and reduced limb morbidity (tissue necrosis, bone re-breakage, scabbing, reopening of wounds, etc.) in animals that received engineered muscle treatments.
Beyond surgical reconstruction, healing outcomes can be controlled through rehabilitation. Physical therapy improves range of motion, gait, mobility and reduces muscular atrophy, joint stiffness, and risk of venous thromboembolism. Although rehabilitation is broadly prescribed, there remains a knowledge gap in how to synergize surgical reconstructive therapies with appropriate rehabilitation regimens. However, our group has designed nanoscale materials that synergistically work with exercise stimulation to improve healing following muscle trauma. We have shown that cell-free nano-patterned scaffolds improved muscle vascularization and innervation of a mouse VML injury when combined with running exercise.
Taken together our collective studies demonstrate control over musculoskeletal recovery through the multi-scale integration of biophysical and biomechanical cues using regenerative engineering and exercise rehabilitation.
Acknowledgements (Optional): : Funding support: NIH/NIAMS (R01AR080150); NIH/NHLBI (R00HL136701); Collins Medical Trust; MTF Biologics; OHSU Medical Research Foundation; Alliance for Regenerative Rehabilitation Research and Training (CNVA00048860); National Science Foundation (DGE-1937961)
