PuraStat RADA16 Self-Assembling Peptide Reduces Postoperative Abdominal Adhesion Formation in a Rabbit Cecal Sidewall Injury Model.

Objective: To evaluate the effect of PuraStat (2.5% RADA16) administration on postoperative abdominal adhesion formation in an in vivo model. Methods: Anesthetized New Zealand white rabbits underwent cecal sidewall abrasion surgery in which the cecal serosa and juxtaposed parietal peritoneum were abraded after access through an abdominal midline incision. Eight animals were randomized to receive PuraStat administration at the interface of the injured tissues before incision closure, and five animals served as untreated controls. Treated animals received 3-12 ml PuraStat solution per lesion. Animals were sacrificed 14 days after surgery and examined for adhesion formation at the wound site. Results: At study terminus, adhesions were identified in 90% (9/10) of abraded cecum/peritoneal wound sites in untreated controls versus 25% (4/16) of PuraStat-treated sites (p = 0.004). Mean ± SD Total Adhesion Score (average of the values for extent + strength of the adhesion in both defects per animal; maximum score = 14 points) was significantly 76% lower in PuraStat-treated animals (2.0 ± 3.0 points) compared to untreated controls (8.2 ± 1.9 points) (p = 0.029). Mean adhesion coverage area of wound sites was 79% lower in PuraStat-treated animals than controls (p < 0.001), and mean adhesion durability was 72% lower in PuraStat-treated animals versus controls (p = 0.005). Remnant hydrogel was observed at the wound sites of 75% of treated animals at postoperative Day 14. Conclusion: PuraStat treatment has a positive protective effect in the cecal sidewall injury model, and significantly reduces abdominal adhesion formation at the interface of the injured cecum and overlying peritoneal sidewall defect.

Clinical Use of the Self-Assembling Peptide RADA16: A Review of Current and Future Trends in Biomedicine.

RADA16 is a synthetic peptide that exists as a viscous solution in an acidic formulation. In an acidic aqueous environment, the peptides spontaneously self-assemble into ß-sheet nanofibers. Upon exposure and buffering of RADA16 solution to the physiological pH of biological fluids such as blood, interstitial fluid and lymph, the nanofibers begin physically crosslinking within seconds into a stable interwoven transparent hydrogel 3-D matrix. The RADA16 nanofiber hydrogel structure closely resembles the 3-dimensional architecture of native extracellular matrices. These properties make RADA16 formulations ideal topical hemostatic agents for controlling bleeding during surgery and to prevent post-operative rebleeding. A commercial RADA16 formulation is currently used for hemostasis in cardiovascular, gastrointestinal, and otorhinolaryngological surgical procedures, and studies are underway to investigate its use in wound healing and adhesion reduction. Straightforward application of viscous RADA16 into areas that are not easily accessible circumvents technical challenges in difficult-to-reach bleeding sites. The transparent hydrogel allows clear visualization of the surgical field and facilitates suture line assessment and revision. The shear-thinning and thixotropic properties of RADA16 allow its easy application through a narrow nozzle such as an endoscopic catheter. RADA16 hydrogels can fill tissue voids and do not swell so can be safely used in close proximity to pressure-sensitive tissues and in enclosed non-expandable regions. By definition, the synthetic peptide avoids potential microbiological contamination and immune responses that may occur with animal-, plant-, or mineral-derived topical hemostats. In vitro experiments, animal studies, and recent clinical experiences suggest that RADA16 nanofibrous hydrogels can act as surrogate extracellular matrices that support cellular behavior and interactions essential for wound healing and for tissue regenerative applications. In the future, the unique nature of RADA16 may also allow us to use it as a depot for precisely regulated drug and biopharmaceutical delivery.

Biodegradable silk catheters for the delivery of therapeutics across anatomical repair sites.

Biodegradable silk catheters for the delivery of therapeutics are designed with a focus on creating porous gradients that can direct the release of molecules away from the implantation site. Though suitable for a range of applications, these catheters are designed for drug delivery to transplanted adipose tissue in patients having undergone a fat grafting procedure. A common complication for fat grafts is the rapid reabsorption of large volume adipose transplants. In order to prolong volume retention, biodegradable catheters can be embedded into transplanted tissue to deliver nutrients, growth factors or therapeutics to improve adipocyte viability, proliferation, and ultimately extend volume retention. Two fabrication methods are developed: a silk gel-spinning technique, which uses a novel flash-freezing step to induce high porosity throughout the bulk of the tube, and a dip-coating process using silk protein solutions doped with a water soluble porogen. Increased porosity aids in the diffusion of drug through the silk tube in a controllable way. Additionally, we interface the porous tubes with ALZET osmotic pumps for implantation into a subcutaneous nude mouse model. The work described herein will discuss the processing parameters as well as the interfacing between pump and cargo therapeutic and the resulting release profiles. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 501-510, 2019.

Acellular bi-layer silk fibroin scaffolds support tissue regeneration in a rabbit model of onlay urethroplasty.

Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a rabbit model of urethra repair. A bi-layer silk fibroin matrix was fabricated by a solvent-casting/salt leaching process in combination with silk fibroin film casting to generate porous foams buttressed by homogeneous silk fibroin films. Ventral onlay urethroplasty was performed with silk fibroin grafts (Group 1, N = 4) (Width × Length, 1 × 2 cm(2)) in adult male rabbits for 3 m of implantation. Parallel control groups consisted of animals receiving small intestinal submucosa (SIS) implants (Group 2, N = 4) or urethrotomy alone (Group 3, N = 3). Animals in all groups exhibited 100% survival prior to scheduled euthanasia and achieved voluntary voiding following 7 d of initial catheterization. Retrograde urethrography of each implant group at 3 m post-op revealed wide urethral calibers and preservation of organ continuity similar to pre-operative and urethrotomy controls with no evidence of contrast extravasation, strictures, fistulas, or stone formation. Histological (hematoxylin and eosin and Masson's trichrome), immunohistochemical, and histomorphometric analyses demonstrated that both silk fibroin and SIS scaffolds promoted similar extents of smooth muscle and epithelial tissue regeneration throughout the original defect sites with prominent contractile protein (α-smooth muscle actin and SM22α) and cytokeratin expression, respectively. De novo innervation and vascularization were also evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Following 3 m post-op, minimal acute inflammatory reactions were elicited by silk fibroin scaffolds characterized by the presence of eosinophil granulocytes while SIS matrices promoted chronic inflammatory responses indicated by mobilization of mononuclear cell infiltrates. The results of this study demonstrate that bi-layer silk fibroin scaffolds represent promising biomaterials for onlay urethroplasty, capable of promoting similar degrees of tissue regeneration in comparison to conventional SIS scaffolds, but with reduced immunogenicity.

All-water-based electron-beam lithography using silk as a resist.

Traditional nanofabrication techniques often require complex lithographic steps and the use of toxic chemicals. To move from the laboratory scale to large scales, nanofabrication should be carried out using alternative procedures that are simple, inexpensive and use non-toxic solvents. Recent efforts have focused on nanoimprinting and the use of organic resists (such as quantum dot-polymer hybrids, DNA and poly(ethylene glycol)), which still require, for the most part, noxious chemicals for processing. Significant advances have been achieved using 'green' resists that can be developed with water, but so far these approaches have suffered from low electron sensitivity, line edge roughness and scalability constraints. Here, we present the use of silk as a natural and biofunctional resist for electron-beam lithography. The process is entirely water-based, starting with the silk aqueous solution and ending with simple development of the exposed silk film in water. Because of its polymorphic crystalline structure, silk can be used either as a positive or negative resist through interactions with an electron beam. Moreover, silk can be easily modified, thereby enabling a variety of 'functional resists', including biologically active versions. As a proof of principle of the viability of all-water-based silk electron-beam lithography (EBL), we fabricate nanoscale photonic lattices using both neat silk and silk doped with quantum dots, green fluorescent proteins (GFPs) or horseradish peroxidase (HRP).

Impact of sterilization on the enzymatic degradation and mechanical properties of silk biomaterials.

The effect of some sterilization methods (autoclaving and ethanol treatments) on the degradation rate and mechanical properties of two types of porous silk scaffolds (aqueous- and hexafluoroisopropanol-derived) is evaluated. Changes in secondary structure, crystal size, and supramolecular features of silk fibroin, resulting from sterilization, are tracked to elucidate molecular level effects on protease XIV enzymatic degradation and compressive mechanical properties. The structural features and pore sizes of the silk scaffolds remain intact after both sterilization processes. Autoclave sterilization dramatically reduce the degradation rate of the silk scaffolds in response to protease XIV and significantly increase mechanical properties, in contrast to scaffolds sterilized with 70% ethanol. Higher ß-sheet content and larger crystal size are observed after autoclaving, unlike in response to 70% ethanol sterilization, based on examination of Fourier transform (FT) IR spectroscopy and wide-angle X-ray scattering (WAXS). In addition, thermal analysis finds supramolecular features within silk fibroin amorphous regions, including the glass transition temperature (Tg ), heat capacity of glass transition (ΔCp-Tg ), and thermal gravimetric degradability. Such supramolecular level changes are related to the shift in enzymatic degradation and mechanical properties due to autoclaving versus treatment with 70% EtOH. The changes in supramolecular organization in amorphous regions can retard enzyme diffusion through the glassy regions of the silk matrix or/and hinder binding of enzymes, while also stiffening these matrices.

Bladder tissue regeneration using acellular bi-layer silk scaffolds in a large animal model of augmentation cystoplasty.

Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6 × 6 cm(2)) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded nonsurgical control gains (144%) encountered due to animal growth.In addition, animals augmented with both matrix configurations displayed increases in bladder compliance over pre-operative levels(Group 1: 357%; Group 2: 338%) similar to growth-related elevations observed in non-surgical controls (354%) [corrected]. Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,ß-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair.

Villification: how the gut gets its villi.

The villi of the human and chick gut are formed in similar stepwise progressions, wherein the mesenchyme and attached epithelium first fold into longitudinal ridges, then a zigzag pattern, and lastly individual villi. We find that these steps of villification depend on the sequential differentiation of the distinct smooth muscle layers of the gut, which restrict the expansion of the growing endoderm and mesenchyme, generating compressive stresses that lead to their buckling and folding. A quantitative computational model, incorporating measured properties of the developing gut, recapitulates the morphological patterns seen during villification in a variety of species. These results provide a mechanistic understanding of the formation of these elaborations of the lining of the gut, essential for providing sufficient surface area for nutrient absorption.

Multiple silk coatings on biphasic calcium phosphate scaffolds: effect on physical and mechanical properties and in vitro osteogenic response of human mesenchymal stem cells.

Ceramic scaffolds such as biphasic calcium phosphate (BCP) have been widely studied and used for bone regeneration, but their brittleness and low mechanical strength are major drawbacks. We report the first systematic study on the effect of silk coating in improving the mechanical and biological properties of BCP scaffolds, including (1) optimization of the silk coating process by investigating multiple coatings, and (2) in vitro evaluation of the osteogenic response of human mesenchymal stem cells (hMSCs) on the coated scaffolds. Our results show that multiple silk coatings on BCP ceramic scaffolds can achieve a significant coating effect to approach the mechanical properties of native bone tissue and positively influence osteogenesis by hMSCs over an extended period. The silk coating method developed in this study represents a simple yet effective means of reinforcement that can be applied to other types of ceramic scaffolds with similar microstructure to improve osteogenic outcomes.

The performance of silk scaffolds in a rat model of augmentation cystoplasty.

The diverse processing plasticity of silk-based biomaterials offers a versatile platform for understanding the impact of structural and mechanical matrix properties on bladder regenerative processes. Three distinct groups of 3-D matrices were fabricated from aqueous solutions of Bombyx mori silk fibroin either by a gel spinning technique (GS1 and GS2 groups) or a solvent-casting/salt-leaching method in combination with silk film casting (FF group). SEM analyses revealed that GS1 matrices consisted of smooth, compact multi-laminates of parallel-oriented silk fibers while GS2 scaffolds were composed of porous (pore size range, 5-50 µm) lamellar-like sheets buttressed by a dense outer layer. Bi-layer FF scaffolds were comprised of porous foams (pore size, ~400 µm) fused on their external face with a homogenous, nonporous silk film. Silk groups and small intestinal submucosa (SIS) matrices were evaluated in a rat model of augmentation cystoplasty for 10 weeks of implantation and compared to cystotomy controls. Gross tissue evaluations revealed the presence of intra-luminal stones in all experimental groups. The incidence and size of urinary calculi was the highest in animals implanted with gel spun silk matrices and SIS with frequencies ≥57% and stone diameters of 3-4 mm. In contrast, rats augmented with FF scaffolds displayed substantially lower rates (20%) and stone size (2 mm), similar to the levels observed in controls (13%, 2 mm). Histological (hematoxylin and eosin, Masson's trichrome) and immunohistochemical (IHC) analyses showed comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites supported by all matrix groups similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent uroplakin and p63 protein expression in all experimental groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by Fox3-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. In comparison to other biomaterial groups, cystometric analyses at 10 weeks post-op revealed that animals implanted with the FF matrix configuration displayed superior urodynamic characteristics including compliance, functional capacity, as well as spontaneous non voiding contractions consistent with control levels. Our data demonstrate that variations in scaffold processing techniques can influence the in vivo functional performance of silk matrices in bladder reconstructive procedures.

Evaluation of silk biomaterials in combination with extracellular matrix coatings for bladder tissue engineering with primary and pluripotent cells.

Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFß1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results demonstrate that silk scaffolds support primary and pluripotent cell responses pertinent to bladder tissue engineering and that scaffold morphology and fibronectin coatings influence these processes.

A statistical algorithm for assessing cellular alignment.

Current statistical techniques for analyzing cellular alignment data in the fields of biomaterials and tissue engineering are limited because of heuristic and less quantitative approaches. For example, generally a cutoff degree limit (commonly 20 degrees) is arbitrarily defined within which cells are considered "aligned." The effectiveness of a patterned biomaterial in guiding the alignment of cells, such as neurons, is often critical to predict relationships between the biomaterial design and biological outcomes, both in vitro and in vivo. This becomes particularly important in the case of peripheral neurons, which require precise axon guidance to obtain successful regenerative outcomes. To address this issue, we have developed a protocol for processing cellular alignment data sets, which implicitly determines an "angle of alignment." This was accomplished as follows: cells "aligning" with an underlying, anisotropic scaffold display uniformly distributed angles up to a cutoff point determined by how effective the biomaterial is in aligning cells. Therefore, this fact was then used to determine where an alignment angle data set diverges from a uniform distribution. This was accomplished by measuring the spacing between the collected, increasingly ordered angles and analyzing their underlying distributions using a normalized cumulative periodogram criterion. The proposed protocol offers a novel way to implicitly define cellular alignment, with respect to various anisotropic biomaterials. This method may also offer an alternative to assess cellular alignment, which could offer improved predictive measures related to biological outcomes. Furthermore, the approach described can be used for a broad range of cell types grown on 2D surfaces, but would not be applicable to 3D scaffold systems in the present format.

Laminar silk scaffolds for aligned tissue fabrication.

3D-biomaterial scaffolds with aligned architecture are of vital importance in tissue regeneration. A generic method is demonstrated to produce aligned biomaterial scaffolds using the physics of directional ice freezing. Homogeneously aligned 3D silk scaffolds with high porosity and alignment are prepared. The method can be adapted to a wide range of polymers and is devoid of any chemical reactions, thus avoiding potential complications associated with by-products. Mechanical properties and cellular responses with chondrocytes and bone-marrow-derived hMSCs are studied, assessing survival, proliferation, and differentiation. In vivo tests suggest biocompatibility of the matrices for future tissue engineering applications, specifically in areas where high cellular alignment is needed.

Functionalized silk biomaterials for wound healing.

Silk protein-biomaterial wound dressings with epidermal growth factor (EGF) and silver sulfadiazine were studied with a cutaneous excisional mouse wound model. Three different material designs and two different drug incorporation techniques were studied to compare wound healing responses. Material formats included silk films, lamellar porous silk films and electrospun silk nanofibers, each studied with the silk matrix alone and with drug loading or drug coatings on the silk matrices. Changes in wound size and histological assessments of wound tissues showed that the functionalized silk biomaterial wound dressings increased wound healing rate, including reepithelialization, dermis proliferation, collagen synthesis and reduced scar formation, when compared to air-permeable Tegaderm tape (3M) (- control) and a commercial wound dressing, Tegaderm Hydrocolloid dressing (3M) (+ control). All silk biomaterials were effective for wound healing, while the lamellar porous films and electrospun nanofibers and the incorporation of EGF/silver sulfadiazine, via drug loading or coating, provided the most rapid wound healing responses. This systematic approach to evaluating functionalized silk biomaterial wound dressings demonstrates a useful strategy to select formulations for further study towards new treatment options for chronic wounds.

A silk-based scaffold platform with tunable architecture for engineering critically-sized tissue constructs.

In the field of tissue engineering and regenerative medicine there is significant unmet need for critically-sized, fully degradable biomaterial scaffold systems with tunable properties for optimizing tissue formation in vitro and tissue regeneration in vivo. To address this need, we have developed a silk-based scaffold platform that has tunable material properties, including localized and bioactive functionalization, degradation rate, and mechanical properties and that provides arrays of linear hollow channels for delivery of oxygen and nutrients throughout the scaffold bulk. The scaffolds can be assembled with dimensions that range from millimeters to centimeters, addressing the need for a critically-sized platform for tissue formation. We demonstrate that the hollow channel arrays support localized and confluent endothelialization. This new platform offers a unique and versatile tool for engineering 'tailored' scaffolds for a range of tissue engineering and regenerative medicine needs.

ß-cyclodextrin-poly(ß-amino ester) nanoparticles for sustained drug delivery across the blood-brain barrier.

Novel biodegradable polymeric nanoparticles composed of ß-cyclodextrin and poly(ß-amino ester) segments have been developed for sustained drug delivery across the blood-brain barrier (BBB). The nanoparticles have been synthesized by cross-linking ß-cyclodextrin with poly(ß-amino ester) via the Michael addition method. The chemical, physical, and degradation properties of the nanoparticles have been characterized by matrix-assisted laser desoption/ionization time-of-flight, attenuated total reflectance Fourier transform infrared spectroscopy, nuclear magnetic resonance, dynamic light scattering, and atomic force microscopy techniques. Bovine and human brain microvascular endothelial cell monolayers have been constructed as in vitro BBB models. Preliminary results show that the nanoparticles do not affect the integrity of the in vitro BBB models, and the nanoparticles have much higher permeability than dextran control across the in vitro BBB models. Doxorubicin has been loaded into the nanoparticles with a loading efficiency of 86%, and can be released from the nanoparticles for at least one month. The developed ß-cyclodextrin-poly(ß-amino ester) nanoparticles might be useful as drug carriers for transporting drugs across the BBB to treat chronic diseases in the brain.

Patterned silk film scaffolds for aligned lamellar bone tissue engineering.

The alignment and osteogenic differentiation of MSCs on patterned silk films (PF) is investigated as a bottom-up approach toward engineering bone lamellae. Screening PF with various groove dimensions shows that cell alignment is mediated by both the pattern width and depth. MSCs are differentiated in osteogenic medium for four weeks on flat films and on the PF that produce the best alignment. The PF support osteogenic differentiation while also inducing lamellar alignment of cells and matrix deposition. A secondary alignment effect is noted on the PF where a new layer of aligned cells grows over the first layer, but rotated obliquely to the underlying pattern. This layering and rotation of the MSCs resembles the cellular organization observed in native lamellar bone.

Evaluation of biomaterials for bladder augmentation using cystometric analyses in various rodent models.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity(1). Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures(2). Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails(3). However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy(4,5). Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic(6,7). However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis(8), graft contracture(9), and calcification(10), thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed. In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.(11,12) Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders(11,12). Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model(11,12). These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

High-strength silk protein scaffolds for bone repair.

Biomaterials for bone tissue regeneration represent a major focus of orthopedic research. However, only a handful of polymeric biomaterials are utilized today because of their failure to address critical issues like compressive strength for load-bearing bone grafts. In this study development of a high compressive strength (~13 MPa hydrated state) polymeric bone composite materials is reported, based on silk protein-protein interfacial bonding. Micron-sized silk fibers (10-600 µm) obtained utilizing alkali hydrolysis were used as reinforcement in a compact fiber composite with tunable compressive strength, surface roughness, and porosity based on the fiber length included. A combination of surface roughness, porosity, and scaffold stiffness favored human bone marrow-derived mesenchymal stem cell differentiation toward bone-like tissue in vitro based on biochemical and gene expression for bone markers. Further, minimal in vivo immunomodulatory responses suggested compatibility of the fabricated silk-fiber-reinforced composite matrices for bone engineering applications.

Annulus fibrosus tissue engineering using lamellar silk scaffolds.

Degeneration of the intervertebral disc (IVD) represents a significant muscular skeletal disease. Recently, scaffolds composed of synthetic, natural and hybrid biomaterials have been investigated as options to restore the IVD; however, they lack the hallmark lamellar morphological features of annulus fibrosus (AF) tissue. The goal of regenerating the disc is to achieve anatomical morphology as well as restoration of mechanical and biological function. In this study, two types of scaffold morphology formed from silk fibroin were investigated towards the goal of AF tissue restoration. The first design mimics the lamellar features of the IVD that are associated with the AF region. The second is a porous spongy scaffold that serves as a control. Toroidal scaffolds were formed from the lamellar and porous silk material systems to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm and a height of 3 mm. The inter-lamellar spacing in the lamellar scaffold was 150-250 µm and the average pore sizes in the porous scaffolds were 100-250 µm. The scaffolds were seeded with porcine AF cells and, after growth over defined time frames in vitro, histology, biochemical assays, mechanical testing and gene expression indicated that the lamellar scaffold generated results that were more favourable in terms of ECM expression and tissue function than the porous scaffold for AF tissue. Further, the seeded porcine AF cells supported the native shape of AF tissue in the lamellar silk scaffolds. The lamellar silk scaffolds were effective in the formation of AF-like tissue in vitro.