Development of systemic immune dysregulation in a rat trauma model of biomaterial-associated infection.

Orthopedic biomaterial-associated infections remain a major clinical challenge, with Staphylococcus aureus being the most common pathogen. S. aureus biofilm formation enhances immune evasion and antibiotic resistance, resulting in a local, indolent infection that can persist long-term without symptoms before eventual hardware failure, bone non-union, or sepsis. Immune modulation is an emerging strategy to combat host immune evasion by S. aureus. However, most immune modulation strategies are focused on local immune responses at the site of infection, with little emphasis on understanding the infection-induced and orthopedic-related systemic immune responses of the host, and their role in local infection clearance and tissue regeneration. This study utilized a rat bone defect model to investigate how implant-associated infection affects the systemic immune response. Long-term systemic immune dysregulation was observed with a significant systemic decrease in T cells and a concomitant increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) compared to non-infected controls. Further, the control group exhibited a regulated and coordinated systemic cytokine response, which was absent in the infection group. Multivariate analysis revealed high levels of MDSCs to be most correlated with the infection group, while high levels of T cells were most correlated with the control group. Locally, the infection group had attenuated macrophage infiltration and increased levels of MDSCs in the local soft tissue compared to non-infected controls. These data reveal the widespread impacts of an orthopedic infection on both the local and the systemic immune responses, uncovering promising targets for diagnostics and immunotherapies that could optimize treatment strategies and ultimately improve patient outcomes.

Development of a Small Animal Ankle Arthrodesis Model.

BACKGROUND: Our understanding of the biology of ankle arthrodesis is based largely on work in spine and long bone animal models. However, the local soft tissue and vascular anatomy of the foot and ankle is different from that of the spine. Accordingly, the objective of this study was to develop a small animal ankle arthrodesis model. METHODS: A total of 12 Lewis rats successfully underwent ankle arthrodesis with stabilization consisting of a single Kirschner wire across the prepared tibiotalar joint. Based on high nonunion rates with this initial procedure, a modification was made consisting of a second pin crossing the joint. A total of 6 rats underwent the second procedure. Radiographs were taken postoperatively and in 2-week intervals up to 10 weeks. Micro computed tomography (µCT) and histological analysis was conducted at 10 weeks to assess the fusion mass. Osseous bridging of greater than 50% across the tibiotalar joint was deemed a successful fusion. RESULTS: µCT analysis determined that 11 of the 12 rats in the single-pin cohort developed nonunions (8.3% fusion rate). In the dual-pin cohort, all 6 animals successfully fused (100% fusion rate). Histological analysis supported the radiographic imaging conclusions. CONCLUSION: While the initial procedure had a high nonunion rate, enhancing the stability of the fixation greatly increased the union rate. CLINICAL RELEVANCE: The present work demonstrates the first reliable small animal ankle arthrodesis model. We believe that this model can be used in the development of novel therapies aimed at decreasing complications and increasing fusion rates.

Biological evaluation and finite-element modeling of porous poly(para-phenylene) for orthopaedic implants.

Poly(para-phenylene) (PPP) is a novel aromatic polymer with higher strength and stiffness than polyetheretherketone (PEEK), the gold standard material for polymeric load-bearing orthopaedic implants. The amorphous structure of PPP makes it relatively straightforward to manufacture different architectures, while maintaining mechanical properties. PPP is promising as a potential orthopaedic material; however, the biocompatibility and osseointegration have not been well investigated. The objective of this study was to evaluate biological and mechanical behavior of PPP, with or without porosity, in comparison to PEEK. We examined four specific constructs: 1) solid PPP, 2) solid PEEK, 3) porous PPP and 4) porous PEEK. Pre-osteoblasts (MC3T3) exhibited similar cell proliferation among the materials. Osteogenic potential was significantly increased in the porous PPP scaffold as assessed by ALP activity and calcium mineralization. In vivo osseointegration was assessed by implanting the cylindrical materials into a defect in the metaphysis region of rat tibiae. Significantly more mineral ingrowth was observed in both porous scaffolds compared to the solid scaffolds, and porous PPP had a further increase compared to porous PEEK. Additionally, porous PPP implants showed bone formation throughout the porous structure when observed via histology. A computational simulation of mechanical push-out strength showed approximately 50% higher interfacial strength in the porous PPP implants compared to the porous PEEK implants and similar stress dissipation. These data demonstrate the potential utility of PPP for orthopaedic applications and show improved osseointegration when compared to the currently available polymeric material. STATEMENT OF SIGNIFICANCE: PEEK has been widely used in orthopaedic surgery; however, the ability to utilize PEEK for advanced fabrication methods, such as 3D printing and tailored porosity, remain challenging. We present a promising new orthopaedic biomaterial, Poly(para-phenylene) (PPP), which is a novel class of aromatic polymers with higher strength and stiffness than polyetheretherketone (PEEK). PPP has exceptional mechanical strength and stiffness due to its repeating aromatic rings that provide strong anti-rotational biaryl bonds. Furthermore, PPP has an amorphous structure making it relatively easier to manufacture (via molding or solvent-casting techniques) into different geometries with and without porosity. This ability to manufacture different architectures and use different processes while maintaining mechanical properties makes PPP a very promising potential orthopaedic biomaterial which may allow for closer matching of mechanical properties between the host bone tissue while also allowing for enhanced osseointegration. In this manuscript, we look at the potential of porous and solid PPP in comparison to PEEK. We measured the mechanical properties of PPP and PEEK scaffolds, tested these scaffolds in vitro for osteocompatibility with MC3T3 cells, and then tested the osseointegration and subsequent functional integration in vivo in a metaphyseal drill hole model in rat tibia. We found that PPP permits cell adhesion, growth, and mineralization in vitro. In vivo it was found that porous PPP significantly enhanced mineralization into the construct and increased the mechanical strength required to push out the scaffold in comparison to PEEK. This is the first study to investigate the performance of PPP as an orthopaedic biomaterial in vivo. PPP is an attractive material for orthopaedic implants due to the ease of manufacturing and superior mechanical strength.

A Novel Technique for Accelerated Culture of Murine Mesenchymal Stem Cells that Allows for Sustained Multipotency.

Bone marrow derived mesenchymal stem cells (MSCs) are regularly utilized for translational therapeutic strategies including cell therapy, tissue engineering, and regenerative medicine and are frequently used in preclinical mouse models for both mechanistic studies and screening of new cell based therapies. Current methods to culture murine MSCs (mMSCs) select for rapidly dividing colonies and require long-term expansion. These methods thus require months of culture to generate sufficient cell numbers for feasibility studies in a lab setting and the cell populations often have reduced proliferation and differentiation potential, or have become immortalized cells. Here we describe a simple and reproducible method to generate mMSCs by utilizing hypoxia and basic fibroblast growth factor supplementation. Cells produced using these conditions were generated 2.8 times faster than under traditional methods and the mMSCs showed decreased senescence and maintained their multipotency and differentiation potential until passage 11 and beyond. Our method for mMSC isolation and expansion will significantly improve the utility of this critical cell source in pre-clinical studies for the investigation of MSC mechanisms, therapies, and cell manufacturing strategies.

Electrospun vascular scaffold for cellularized small diameter blood vessels: A preclinical large animal study.

The strategy of vascular tissue engineering is to create a vascular substitute by combining autologous vascular cells with a tubular-shaped biodegradable scaffold. We have previously developed a novel electrospun bilayered vascular scaffold that provides proper biological and biomechanical properties as well as structural configuration. In this study, we investigated the clinical feasibility of a cellularized vascular scaffold in a preclinical large animal model. We fabricated the cellularized vascular construct with autologous endothelial progenitor cell (EPC)-derived endothelial cells (ECs) and smooth muscle cells (SMCs) followed by a pulsatile bioreactor preconditioning. This fully cellularized vascular construct was tested in a sheep carotid arterial interposition model. After preconditioning, confluent and mature EC and SMC layers in the scaffold were achieved. The cellularized constructs sustained the structural integrity with a high degree of graft patency without eliciting an inflammatory response over the course of the 6-month period in sheep. Moreover, the matured EC coverage on the lumen and a thick smooth muscle layer were formed at 6months after transplantation. We demonstrated that electrospun bilayered vascular scaffolds in conjunction with autologous vascular cells may be a clinically applicable alternative to traditional prosthetic vascular graft substitutes. STATEMENT OF SIGNIFICANCE: This study demonstrates the utility of tissue engineering to provide platform technologies for rehabilitation of patients recovering from severe, devastating cardiovascular diseases. The long-term goal is to provide alternatives to vascular grafting using bioengineered blood vessels derived from an autologous cell source with a functionalized vascular scaffold. This novel bilayered vascular construct for engineering blood vessels is designed to offer "off-the-shelf" availability for clinical translation.

Engineered small diameter vascular grafts by combining cell sheet engineering and electrospinning technology.

Tissue engineering offers an attractive approach to creating functional small-diameter (<5mm) blood vessels by combining autologous cells with a natural and/or synthetic scaffold under suitable culture conditions, which results in a tubular construct that can be implanted in vivo. We have previously developed a vascular scaffold fabricated by electrospinning poly(ε-caprolactone) (PCL) and type I collagen that mimics the structural and biomechanical properties of native vessels. In this study, we investigated whether a smooth muscle cell (SMC) sheet could be combined with the electrospun vascular scaffolds to produce a more mature smooth muscle layer as compared to the conventional cell seeding method. The pre-fabricated SMC sheet, wrapped around the vascular scaffold, provided high cell seeding efficiency (approx. 100%) and a mature smooth muscle layer that expressed strong cell-to-cell junction, connexin 43 (CX43), and contractile proteins, α smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK). Moreover, bioreactor-associated preconditioning of the SMC sheet-combined vascular scaffold maintained high cell viability (95.9 ± 2.7%) and phenotypes and improved cellular infiltration and mechanical properties (35.7% of tensile strength, 47.5% of elasticity, and 113.2% of elongation at break).