Controlled pore anisotropy in chitosan-gelatin cryogels for use in bone tissue engineering.

In tissue engineering, the development of an appropriate scaffold is crucial to provide a framework for new tissue growth. The use of cryogels as scaffolds shows promise due to their macroporous structure, but the pore size, distribution, and interconnectivity is highly variable depending on the fabrication process. The objective of the current research is to provide a technique for controlled anisotropy in chitosan-gelatin cryogels to develop scaffolds for bone tissue engineering application. A mold was developed using additive manufacturing to be used during the freezing process in order to fabricate cryogels with a more interconnected pore structure. The scaffolds were tested to evaluate their porosity, mechanical strength, and to observe cell infiltration through the cryogel. It was found that the use of the mold allowed for the creation of designated pores within the cryogel structure which facilitated cell infiltration to the center of the scaffold without sacrificing mechanical integrity of the structure.

Sexual differences in bone porosity, osteocyte density, and extracellular matrix organization due to osteoblastic-specific Bmp2 deficiency in mice.

Clinical studies have come to conflicting conclusions regarding BMP2 deficiency's link to regulating bone mass and increasing fracture risk. This may be due to the signaling protein having sex- or age-dependent effects. Previous pre-clinical studies have supported a role, but have not adequately determined the physical mechanism causing altered bulk material properties. This study investigated the physical effects of Bmp2 ablation from osteogenic lineage cells (Osx-Cre; Bmp2fl/fl) in 10- and 15-week-old male and female mice. Bones collected post-mortem were subjected to fracture toughness testing, reference point indentation testing, microCT, and histological analysis to determine the multi-scale relationships between mechanical/material behavior and collagen production, collagen organization, and bone architecture. BMP2-deficient bones were smaller, more brittle, and contained more lacunae-scale voids and cortical pores. The cellular density was significantly increased and there were material-level differences measured by reference point indentation, independently of collagen fiber alignment or organization. The disparities in bone size and in bone fracture toughness between genotypes were especially striking in males at 15-weeks-old. Together, this study suggests that there are sex- and age-dependent effects of BMP2 deficiency. The results from both sexes also warrant further investigation into BMP2 deficiency's role in osteoblasts' transition to osteocytes and overall bone porosity.

Masquelet's induced membrane technique: Review of current concepts and future directions.

Masquelet's induced membrane technique (MIMT) is a relatively new, two-stage surgical procedure to reconstruct segmental bone defects. First performed by Dr. Masquelet in the mid-1980s, MIMT has shown great promise to revolutionize critical-sized bone defect repair and has several advantages over its alternative, distraction osteogenesis (DO). Also, its success in extremely challenging cases (defects > 15 cm) suggests that its study could lead to discovery of novel biological mechanisms that might be at play during segmental defect healing and fracture non-union. MIMT's advantages over DO have led to a world-wide increase in MIMT procedures over the past decades. However, MIMT often needs to be repeated and so the average initial success rate in adults lags significantly behind that of DO (86% vs 95%, respectively). The autologous foreign-body membrane created during the first stage by the immune system's response to a polymethyl methacrylate bone cement spacer is critical to supporting the morselized bone graft implanted in the second stage. However, the biological and/or physical mechanisms by which the membrane supports graft to bone union are unclear. This lack of knowledge makes refining MIMT and improving the success rates through technique improvements and patient selection a significant challenge and hinders wider adoption. In this review, current knowledge from basic, translational, and clinical studies is summarized. The dynamics of both stages under normal conditions as well as with drug or material perturbations is discussed along with perspectives on high-priority future research directions.

MicroCT vascular network analysis program: Development, validation, and comparison to manufacturer software.

The dependence on angiogenesis for bone repair makes accurate measuring of vascular networks of great importance to orthopedic researchers. A three-dimensional imaging modality like microcomputed tomography (µCT) would better capture these networks than histology. There are commercially available programs to analyze vessel networks in three dimensions, but these may be too costly for laboratories. Alternatively, µCT trabecular software could be used but may not be appropriate. The goal of this project was to develop a vascular network analysis protocol based on freely or commonly available software and compare its performance to that of a µCT trabecular analysis software. The protocol developed, called vascular network analysis or VNA, relies on two modules in Fiji ImageJ and a custom MATLAB program. We validated the software and compared it to a µCT trabecular analysis program (MicroCT) using in silico models of increasing complexity and differing homogeneity. In general, VNA outcomes were significantly different from true values, but most were within an acceptable percent error (<10%). VNA and MicroCT performed almost identically for volume but significantly differently for average vessel diameter. For the homogenous models, the average diameters differed only slightly but were starkly different for the heterogeneous models. In the most heterogeneous system, the MicroCT software overestimated average diameter by about 650% from true. VNA was within 1% of true for the same model. In conclusion, we have developed a program to analyze vascular networks from MicroCT scans which is easily accessible, insensitive to network homogeneity, and of higher accuracy compared to a µCT trabecular analysis software.

Mechanomics Approaches to Understand Cell Behavior in Context of Tissue Neogenesis, During Prenatal Development and Postnatal Healing.

Mechanomics represents the natural progression of knowledge at the intersection of mechanics and biology with the aim to codify the role of mechanical environment on biological adaptation. Compared to the mapping of the human genome, the challenge of mapping the mechanome remains unsolved. Solving this grand challenge will require both top down and bottom up R&D approaches using experimental and computational tools to visualize and measure adaptation as it occurs. Akin to a mechanical test of a smart material that changes its mechanical properties and local environment under load, stem cells adapt their shape, cytoskeletal architecture, intrinsic mechanical properties, as well as their own niche, through cytoskeletal adaptation as well as up- and down-regulation of structural proteins that modulate their mechanical milieux. Recent advances in live cell imaging allow for unprecedented study and measurements of displacements, shape and volume changes in stem cells, reconfiguring of cytoskeletal machinery (nucleus, cytoskeleton), in response to controlled mechanical forces and stresses applied at cellular boundaries. Coupled with multiphysics computational and virtual power theoretical approaches, these novel experimental approaches enable mechanical testing of stem cells, multicellular templates, and tissues inhabited by stem cells, while the stem cells themselves evolve over time. The novel approach is paving the way to decipher mechanisms of structural and functional adaptation of stem cells in response to controlled mechanical cues. This mini-review outlines integrated approaches and methodologies implemented to date in a series of studies carried out by our consortium. The consortium's body of work is described in context of current roadblocks in the field and innovative, breakthrough solutions and is designed to encourage discourse and cross disciplinary collaboration in the scientific community.

Masquelet Technique: Effects of Spacer Material and Micro-topography on Factor Expression and Bone Regeneration.

We and others have shown that changing surface characteristics of the spacer implanted during the first Masquelet stage alters some aspects of membrane development. Previously we demonstrated that titanium (TI) spacers create membranes that are better barriers to movement of solutes > 70 kDa in size than polymethyl methacrylate (PMMA) induced-membranes, and roughening creates more mechanically compliant membranes. However, it is unclear if these alterations affect the membrane's biochemical environment or bone regeneration during the second stage. Ten-week-old, male Sprague-Dawley rats underwent an initial surgery to create an externally stabilized 6 mm femoral defect. PMMA or TI spacers with smooth (~ 1 µm) or roughened (~ 8 µm) surfaces were implanted. Four weeks later, rats were either euthanized for membrane harvest or underwent the second Masquelet surgery. TI spacers induced thicker membranes that were similar in structure and biochemical expression. All membranes were bilayered with the inner layer having increased factor expression [bone morphogenetic protein 2 (BMP2), transforming growth factor beta (TGFß), interleukin 6 (IL6), and vascular endothelial growth factor (VEGF)]. Roughening increased overall IL6 levels. Ten-weeks post-engraftment, PMMA-smooth induced membranes better supported bone regeneration (60% union). The other groups only had 1 or 2 that united (9-22%). There were no significant differences in any micro computed tomography or dynamic histology outcome. In conclusion, this study suggests that the membrane's important function in the Masquelet technique is not simply as a barrier. There is likely a critical biochemical, cellular, or vascular component as well.

Biomimetic sponges for regeneration of skeletal muscle following trauma.

Skeletal muscle is inept in regenerating after traumatic injuries due to significant loss of basal lamina and the resident satellite cells. To improve regeneration of skeletal muscle, we have developed biomimetic sponges composed of collagen, gelatin, and laminin (LM)-111 that were crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Collagen and LM-111 are crucial components of the muscle extracellular matrix and were chosen to impart bioactivity whereas gelatin and EDC were used to provide mechanical strength to the scaffold. Morphological and mechanical evaluation of the sponges showed porous structure, water-retention capacity and a compressive modulus of 590-808 kPa. The biomimetic sponges supported the infiltration and viability of C2 C12 myoblasts over 5 days of culture. The myoblasts produced higher levels of myokines such as VEGF, IL-6, and IGF-1 and showed higher expression of myogenic markers such as MyoD and myogenin on the biomimetic sponges. Biomimetic sponges implanted in a mouse model of volumetric muscle loss (VML) supported satellite, endothelial, and inflammatory cell infiltration but resulted in limited myofiber regeneration at 2 weeks post-injury. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 92-103, 2019.

Masquelet technique: The effect of altering implant material and topography on membrane matrix composition, mechanical and barrier properties in a rat defect model.

The Masquelet technique is a surgical procedure to regenerate segmental bone defects. The two-phase treatment relies on the production of a vascularized foreign-body membrane to support bone grafts over three times larger than the traditional maximum. Historically, the procedure has always utilized a bone cement spacer to evoke membrane production. However, membrane formation can easily be effected by implant surface properties such as material and topology. This study sought to determine if the membrane's mechanical or barrier properties are affected by changing the spacer material to titanium or roughening the surface finish. Ten-week-old, male Sprague Dawley rats were given an externally stabilized, 6 mm femur defect which was filled with a pre-made spacer of bone cement (PMMA) or titanium (TI) with a smooth (∼1 µm) or roughened (∼8 µm) finish. After 4 weeks of implantation, the membranes were harvested, and the matrix composition, tensile mechanics, shrinkage, and barrier function was assessed. Roughening the spacers resulted in significantly more compliant membranes. TI spacers created membranes that inhibited solute transport more. There were no differences between groups in collagen or elastin distribution. This suggests that different membrane characteristics can be created by altering the spacer surface properties. Surgeons may unknowingly effecting membrane formation via bone cement preparation techniques.

Altering spacer material affects bone regeneration in the Masquelet technique in a rat femoral defect.

The Masquelet technique depends on pre-development of a foreign-body membrane to support bone regeneration with grafts over three times larger than the traditional maximum. To date, the procedure has always used spacers made of bone cement, which is the polymer polymethyl methacrylate (PMMA), to induce the foreign-body membrane. This study sought to compare (i) morphology, factor expression, and cellularity in membranes formed by PMMA, titanium, and polyvinyl alcohol sponge (PVA) spacers in the Masquelet milieu and (ii) subsequent bone regeneration in the same groups. Ten-week-old, male Sprague-Dawley rats were given an externally stabilized, 6 mm femur defect, and a pre-made spacer of PMMA, titanium, or PVA was implanted. All animals were given 4 weeks to form a membrane, and those receiving an isograft were given 10 weeks post-implantation to union. All samples were scanned with microCT to measure phase 1 and phase 2 bone formation. Membrane samples were processed for histology to measure membrane morphology, cellularity, and expression of the factors BMP2, TGFß, VEGF, and IL6. PMMA and titanium spacers created almost identical membranes and phase 1 bone. PVA spacers were uniformly infiltrated with tissue and cells and did not form a distinct membrane. There were no quantitative differences in phase 2 bone formation. However, PMMA induced membranes supported functional union in 6 of 7 samples while a majority of titanium and PVA groups failed to achieve the same. Spacer material can alter the membrane enough to disrupt phase 2 bone formation. The membrane's role in bone regeneration is likely more than just as a physical barrier. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

A preliminary in vitro evaluation of the bioactive potential of cryogel scaffolds incorporated with Manuka honey for the treatment of chronic bone infections.

Previous studies have identified honey as an agent in bacterial inhibition and a mediator in lowering the pH at the wound site. Manuka honey (MH), indigenous to New Zealand, contains a Unique Manuka Factor that provides an additional antibacterial agent. While there are many potential benefits to incorporating MH into wounds, there is currently no ideal way to deliver the material to the site of injury. Cryogels are a type of scaffold that possess high porosity, mechanical stability, and a sponge-like consistency. This study uniquely incorporates varying amounts of MH into cryogel scaffolds, utilizing its properties in a sustained release fashion to assist in the overall healing process, while using the cryogel structure as a tissue template. All cryogels were evaluated to determine the effects of MH on porosity, swelling potential, mechanical durability, and cell compatibility. The release of MH was also quantified to evaluate bacterial clearance potential, and the scaffolds were mineralized to replicate native bone. It was determined that a 5% MH silk fibroin cryogel has the potential to inhibit bacterial growth while still maintaining adequate porosity, mechanical properties, and cell infiltration. Such a scaffold would have use in a number of applications, including bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1918-1933, 2018.

A Comparison of Tissue Engineering Scaffolds Incorporated with Manuka Honey of Varying UMF.

Purpose. Manuka honey (MH) is an antibacterial agent specific to the islands of New Zealand containing both hydrogen peroxide and a Unique Manuka Factor (UMF). Although the antibacterial properties of MH have been studied, the effect of varying UMF of MH incorporated into tissue engineered scaffolds have not. Therefore, this study was designed to compare silk fibroin cryogels and electrospun scaffolds incorporated with a 5% MH concentration of various UMF. Methods. Characteristics such as porosity, bacterial clearance and adhesion, and cytotoxicity were compared. Results. Pore diameters for all cryogels were between 51 and 60 µm, while electrospun scaffolds were 10 µm. Cryogels of varying UMF displayed clearance of approximately 0.16 cm for E. coli and S. aureus. In comparison, the electrospun scaffolds clearance ranged between 0.5 and 1 cm. A glucose release of 0.5 mg/mL was observed for the first 24 hours by all scaffolds, regardless of UMF. With respect to cytotoxicity, neither scaffold caused the cell number to drop below 20,000. Conclusions. Overall, when comparing the effects of the various UMF within the two scaffolds, no significant differences were observed. This suggests that the fabricated scaffolds in this study displayed similar bacterial effects regardless of the UMF value.

The calcification potential of cryogel scaffolds incorporated with various forms of hydroxyapatite for bone regeneration.

Cryogels are advantageous scaffolds for bone regeneration applications due to their high mechanical stability and macroporous structure. Anatomically, bone is composed of collagen and hydroxyapatite and during remodeling, these structural components are replaced. However, early forms of mineralization include calcium salts which take up to months to be converted to the desired hydroxyapatite form. Thus, it is beneficial to provide a primary source of hydroxyapatite within the scaffold, expediting the process of mineralization during bone regeneration. In this study, chitosan-gelatin (CG) cryogels were incorporated with various forms of hydroxyapatite to evaluate effects on the standard characteristics of cryogels, as well as the potential for increased mineralization. Testing included the comparison of porosity, swelling, mechanical integrity, cellular infiltration, and mineralization potential between all types of cryogels. The addition of bone char to CG cryogels produced scaffolds with appropriate porosity and interconnectivity. Additionally, the bone char cryogels exhibited an adequate swelling potential, suitable mechanical properties, excellent cell attachment, and increased mineralization. These properties support this cryogel for such an application in tissue engineering.

The fabrication of cryogel scaffolds incorporated with poloxamer 407 for potential use in the regeneration of the nucleus pulposus.

Degeneration of the nucleus pulposus (NP) is the primary cause of back pain in almost 80% of the world population. The current gold standard treatment for a degenerated NP is a spinal fusion surgery which is costly, temporary, and extremely invasive. Research has been moving towards minimally invasive methods to lessen the collateral damage created during surgery. The use of a tissue-engineered scaffold has the potential to promote a healthy and hydrated environment to regenerate the NP. Cryogels are unique polymeric scaffolds composed of a highly connected, macroporous structure, and are capable of maintaining stability under high deformations. For this study, cryogels have been developed using gelatin and poloxamer 407 (P407) at varying ratios to determine the ideal combination of stability, water retention, and pore size. For the application of NP regeneration, a gelatin-P407 cryogel should be both stable and a well hydrated carrier. The cryogels created varied from a 1:1 gelatin to P407 ratio to a 10:1 ratio. The inclusion of P407 in the cryogels resulted in a significant increase in hydrophilicity, ideal pore size for cell infiltration, mechanical stability over 28 days, and cell infiltration after just 21 days. This novel gelatin-P407 composite cryogel has the potential to be a practical alternative to the spinal fusion procedure, saving patients hundreds of thousands of dollars and, ideally, leading to improved patient outcome.

Remnant Woven Bone and Calcified Cartilage in Mouse Bone: Differences between Ages/Sex and Effects on Bone Strength.

INTRODUCTION: Mouse models are used frequently to study effects of bone diseases and genetic determinates of bone strength. Murine bones have an intracortical band of woven bone that is not present in human bones. This band is not obvious under brightfield imaging and not typically analyzed. Due to the band's morphology and location it has been theorized to be remnant bone from early in life. Furthermore, lamellar and woven bone are well known to have differing mechanical strengths. The purpose of this study was to determine (i) if the band is from early life and (ii) if the woven bone or calcified cartilage contained within the band affect whole bone strength. WOVEN BONE ORIGIN STUDIES: In twelve to fourteen week old mice, doxycycline was used to label bone formed prior to 3 weeks old. Doxycycline labeling and woven bone patterns on contralateral femora matched well and encompassed an almost identical cross-sectional area. Also, we highlight for the first time in mice the presence of calcified cartilage exclusively within the band. However, calcified cartilage could not be identified on high resolution cone-beam microCT scans when examined visually or by thresholding methods. MECHANICAL STRENGTH STUDIES: Subsequently, three-point bending was used to analyze the effects of woven bone and calcified cartilage on whole bone mechanics in a cohort of male and female six and 13 week old Balb/C mice. Three-point bending outcomes were correlated with structural and compositional measures using multivariate linear regression. Woven bone composed a higher percent of young bones than older bones. However, calcified cartilage in older bones was twice that of younger bones, which was similar when normalized by area. Area and/or tissue mineral density accounted for >75% of variation for most strength outcomes. Percent calcified cartilage added significant predictive power to maximal force and bending stress. Calcified cartilage and woven bone could have more influence in genetic models where calcified cartilage percent is double our highest value.

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice.

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10-24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.