Katherine R. Hixon

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.

Cryogel scaffolds from patient-specific 3D-printed molds for personalized tissue-engineered bone regeneration in pediatric cleft-craniofacial defects:

Bone defects are extremely common in children with cleft-craniofacial conditions, especially those with alveolar cleft defects and cranial defects. This study used patient-specific 3D-printed molds derived from computed tomography and cryogel scaffold fabrication as a proof of concept for the creation of site-specific implants for bone reconstruction. Cryogel scaffolds are unique tissue-engineered constructs formed at sub-zero temperatures. When thawed, the resulting structure is macroporous, sponge-like, and mechanically durable. Due to these unique properties, cryogels have excellent potential for the treatment of patient-specific bone defects; however, there is little literature on their use in cleft-craniofacial defects. While 3D-printing technology currently lacks the spatial resolution to print the microstructure necessary for bone regeneration, it can be used to create site-specific molds. Thus, it is ideal to integrate these techniques for the fabrication of scaffolds with patient-specific geometry. Overall, all cryogels possessed appropriate geometry to allow for cell infiltration after 28 days. Additionally, suitable mechanical durability was demonstrated where, despite mold geometry, all cryogels could be compressed without exhibiting crack propagation. Such a patient-specific scaffold would be ideal in pediatric cleft-craniofacial defects, as these are complex 3D defects and children have less donor bone availability.

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.

In vitro characterization of MG-63 osteoblast-like cells cultured on organic-inorganic lyophilized gelatin sponges for early bone healing.

The development of three-dimensional porous scaffolds with enhanced osteogenic and angiogenic potential would be beneficial for inducing early-stage bone regeneration. Previous studies have demonstrated the advantages of mineralized and nonmineralized acellular 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linked gelatin sponges enhanced with preparations rich in growth factors, hydroxyapatite, and chitin whiskers. In this study, those same scaffolds were mineralized and dynamically seeded with MG-63 cells. Cell proliferation, protein/cytokine secretion, and compressive mechanical properties of scaffolds were evaluated. It was found that mineralization and the addition of growth factors increased cell proliferation compared to gelatin controls. Cells on all scaffolds responded in an appropriate bone regenerative fashion as shown through osteocalcin secretion and little to no secretion of bone resorbing markers. However, compressive mechanical properties of cellularized scaffolds were not significantly different from acellular scaffolds. The combined results of increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion on scaffolds support the need for the addition of a bone-like mineral surface. Cellularized scaffolds containing growth factors reported similar advantages and mechanical values in the range of native tissues present in the early stages of bone healing. These results suggest that the developed composite sponges exhibited cellular responses and mechanical properties appropriate for promoting early bone healing in various applications.

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.

Tissue Engineering Scaffolds Fabricated in Dissolvable 3D-Printed Molds for Patient-Specific Craniofacial Bone Regeneration

The current gold standard treatment for oral clefts is autologous bone grafting. This treatment, however, presents another wound site for the patient, greater discomfort, and pediatric patients have less bone mass for bone grafting. A potential alternative treatment is the use of tissue engineered scaffolds. Hydrogels are well characterized nanoporous scaffolds and cryogels are mechanically durable, macroporous, sponge-like scaffolds. However, there has been limited research on these scaffolds for cleft craniofacial defects. 3D-printed molds can be combined with cryogel/hydrogel fabrication to create patient-specific tissue engineered scaffolds. By combining 3D-printing technology and scaffold fabrication, we were able to create scaffolds with the geometry of three cleft craniofacial defects. The scaffolds were then characterized to assess the effect of the mold on their physical properties. While the scaffolds were able to completely fill the mold, creating the desired geometry, the overall volumes were smaller than expected. The cryogels possessed porosities ranging from 79.7% to 87.2% and high interconnectivity. Additionally, the cryogels swelled from 400% to almost 1500% of their original dry weight while the hydrogel swelling did not reach 500%, demonstrating the ability to fill a defect site. Overall, despite the complex geometry, the cryogel scaffolds displayed ideal properties for bone reconstruction.

Electrospun biomaterials for dermal regeneration

Abstract The treatment of dermal wounds has evolved significantly throughout the history of mankind, from simplistic dressings and sutures to full regeneration of the skin with the aid of cellularized dressings and dermal regeneration templates. The skin is the largest organ in the body and primarily functions as a barrier to external agents. While the skin has impressive inherent self-renewal/repair abilities, assisted skin regeneration is vital for closure in a number of aberrant wound healing conditions (i.e., burns, pressure ulcers, diabetic ulcers, etc.) and in the reduction of scar formation. Currently, there are several types of nonelectrospun skin grafts on the market, but each possesses inherent disadvantages. Electrospinning is an ideal fabrication method for the construction of the skin due to its nonwoven fibrillar structures, potential for cellular infiltration, attachment, and overall structural support. Electrospun scaffolds also allow for fine control over pore sizes and fiber diameters. With regard to skin applications, both synthetic and natural polymers have been utilized in electrospinning. Synthetic materials have controllable degradation rates and mechanical properties, whereas natural materials are biodegradable, biocompatible, and possess cell attachment sites. The electrospinning process also allows for a number of biomolecules to be readily included within the fibrous network to increase scaffold bioactivity. Additives such as Manuka honey or an assorted cytokine and chemokine milieu can be incorporated into these electrospun scaffolds to promote bacterial inhibition and modify the local inflammatory response, respectively. This review provides an overview of various electrospun scaffold fabrication techniques, scaffold compositions, and dopants to enhance wound healing and regeneration.

Using Electrospun Scaffolds to Promote Macrophage Phenotypic Modulation and Support Wound Healing

Abstract The development of pressure ulcers in spinal cord injury patients is extremely common, often requiring extensive surgical procedures. Macrophages (MACs) play a crucial role in the innate immune system, contributing to wound healing and overall regeneration. MACs have been found to possess the potential to be activated by external factors from their M0 inactive state to an M1 proinflammatory or M2 regenerative state. This study conducted a comprehensive evaluation of MAC phenotype in response to electrospun scaffolds of varying material fiber/pore diameter, fiber stiffness, and +/− inclusion of platelet-rich plasma (PRP). Generally, itwas found that the addition of PRP resulted in decreased pore size, where 5 silk fibroin (SF) had the stiffest fibers. Furthermore, PRP scaffolds demonstrated an increased production of VEGF and chemotaxis. The polycaprolactone (PCL) and SF scaffolds had the largest cell infiltration and proliferation. Overall, it was found that 5% SF had both ideal fiber and pore structure, allowing for cell infiltration further enhanced by the presence of PRP. Additionally, this scaffold led to a reasonable production of VEGF while still allowing fibroblast proliferation to occur. These results suggest that such a scaffold could provide an off-the-shelf product capable of modifying the local MAC response.