Sarah McBride-Gagyi

A comparison of cryogel scaffolds to identify an appropriate structure for promoting bone regeneration

To create an ideal graft substitute for regenerating bone, the scaffold should possess osteoconductive, osteoinductive, and osteogenic properties. Hydrogels are a very common scaffold, but the mechanical integrity and nanoporous structure are not advantageous for bone regeneration. Cryogelation is a technique in which the controlled freezing and thawing of a polymer creates a spongy, macroporous structure with ideal structural characteristics and promising mechanical stability. Hydrogels and cryogels of three different materials (chitosan–gelatin, N-vinyl-2-pyrrolidone, and silk fibroin (SF)) were compared to assess the optimal material and form of scaffold for this application. Cryogel and hydrogel structures were tested in parallel to evaluate porosity, swelling, mechanical integrity, cellular infiltration, and mineralization potential. Cryogels proved superior to hydrogels based on swelling potential and mechanical properties. Among the cryogels, SF demonstrated high pore diameter and area, mineralization upon cellular infiltration, and the largest presence of osteocalcin, a marker of bone formation. These results demonstrate the practicality of cryogels for a bone regeneration application and identify SF as a potential material choice.

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.

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.

Altering spacer material affects bone regeneration in the Masquelet technique in a rat femoral defect: MASQUELET TECHNIQUE: ALTERING SPACER MATERIAL

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, TGFβ, 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 membranes role in bone regeneration is likely more than just as a physical barrier.

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 membranes 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.

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.

Systemically administered MSCs given 24hrs after osteotomy do not affect bone formation in rat distraction osteogenesis

Distraction osteogenesis is a unique postnatal bone formation employed by orthopaedic surgeons to treat many conditions, however, the overall time to external frame removal can be extensive. Any strategies that accelerate healing would improve patient care. Distraction osteogenesis research in the past decade has shown that direct stem cell implantation enhances new bone formation. Systemic implantation would be more clinically desirable. Systemically delivered stem cells have been shown to home to a mandibular distraction site; however, effects on bone formation have not been studied. Ten-week-old, male Sprague-Dawley rats underwent surgery to implant an external fixator-distractor and an osteotomy was performed. Twenty-four hours postoperatively, each rat received tail vein injections of either saline or 10^6 fluorescently labeled primary mesenchymal stem cells. Animals in the validation groups were euthanized two days after surgery and the femora processed for histology. Animals in the experimental groups were given five days of latency, then the femur was lengthened once daily for five days (0.75mm/day, 3.75mm total). Following four weeks of consolidation, the animals were euthanized and the femora were evaluated by microCT and histology to quantify new bone formation. Labeled stem cells were found at the distraction site in validation animals. However, there were no differences in any bone or soft tissue outcomes. Systemic stem cell administration 24 hours after surgery does not improve DO outcomes. It is possible that the animal model was not challenging enough to discriminate any augmentation provided by stem cells.