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.

An Animal Model of Local Breast Cancer Recurrence in the Setting of Autologous Fat Grafting for Breast Reconstruction.

Autologous fat grafting after breast cancer surgery is commonly performed, but concerns about oncologic risk remain. To model the interaction between fat grafting and breast cancer cells, two approaches were employed. In the first approach, graded numbers of viable MDA-MB-231 or BT-474 cells were admixed directly into human fat grafts and injected subcutaneously into immune-deficient mice to determine if the healing graft is a supportive environment for the tumor. In the second approach, graded doses of MDA-MB-231 cells were suspended in Matrigel and injected into the mammary fat pads of mice. Two weeks after the tumor cells engrafted, 100 µL of human adipose tissue was grafted into the same site. Histologically, MDA-MB-231 cells seeded within fat grafts were observed and stained positive for human-specific pan-cytokeratin and Ki67. The BT-474 cells failed to survive when seeded within fat grafts at any dose. In the second approach, MDA-MB-231 cells had a strong trend toward lower Ki67 staining at all doses. Regression analysis on all groups with fat grafts and MDA-MB-231 revealed fat tissue was associated with lower cancer cell Ki67 staining. Healing fat grafts do not support the epithelial BT-474 cell growth, and support the mesenchymal MDA-MB-231 cell growth only at doses ten times greater than in Matrigel controls. Moreover, fat grafts in association with MDA-MB-231 cancer cells already present in the wound resulted in decreased tumor proliferation and increased fibrosis. These findings suggest that clinical fat grafting does not induce breast cancer cell growth, and may even have a suppressive effect. Stem Cells Translational Medicine 2018;7:125-134.

Prevalence of endogenous CD34+ adipose stem cells predicts human fat graft retention in a xenograft model.

BACKGROUND: Fat grafting is a promising technique for soft-tissue augmentation, although graft retention is highly unpredictable and factors that affect graft survival have not been well defined. Because of their capacity for differentiation and growth factor release, adipose-derived stem cells may have a key role in graft healing. The authors' objective was to determine whether biological properties of adipose-derived stem cells present within human fat would correlate with in vivo outcomes of graft volume retention. METHODS: Lipoaspirate from eight human subjects was processed using a standardized centrifugation technique and then injected subcutaneously into the flanks of 6-week-old athymic nude mice. Graft masses and volumes were measured, and histologic evaluation, including CD31+ staining for vessels, was performed 8 weeks after transplantation. Stromal vascular fraction isolated at the time of harvest from each subject was analyzed for surface markers by multiparameter flow cytometry, and also assessed for proliferation, differentiation capacity, and normoxic/hypoxic vascular endothelial growth factor secretion. RESULTS: Wide variation in percentage of CD34+ progenitors within the stromal vascular fraction was noted among subjects and averaged 21.3 ± 15 percent (mean ± SD). Proliferation rates and adipogenic potential among stromal vascular fraction cells demonstrated moderate interpatient variability. In mouse xenograft studies, retention volumes ranged from approximately 36 to 68 percent after 8 weeks, with an overall average of 52 ± 11 percent. A strong correlation (r = 0.78, slope = 0.76, p < 0.05) existed between stromal vascular fraction percentage of CD34+ progenitors and high graft retention. CONCLUSION: Inherent biological differences in adipose tissue exist between patients. In particular, concentration of CD34+ progenitor cells within the stromal vascular fraction may be one of the factors used to predict human fat graft retention.

Functional skeletal muscle formation with a biologic scaffold.

Biologic scaffolds composed of extracellular matrix (ECM) have been used to reinforce or replace damaged or missing musculotendinous tissues in both preclinical studies and in human clinical applications. However, most studies have focused upon morphologic endpoints and few studies have assessed the in-situ functionality of newly formed tissue; especially new skeletal muscle tissue. The objective of the present study was to determine both the in-situ tetanic contractile response and histomorphologic characteristics of skeletal muscle tissue reconstructed using one of four test articles in a rodent abdominal wall model: 1) porcine small intestinal submucosa (SIS)-ECM; 2) carbodiimide-crosslinked porcine SIS-ECM; 3) autologous tissue; or 4) polypropylene mesh. Six months after surgery, the remodeled SIS-ECM showed almost complete replacement by islands and sheets of skeletal muscle, which generated a similar maximal contractile force to native tissue but with greater resistance to fatigue. The autologous tissue graft was replaced by a mixture of collagenous connective tissue, adipose tissue with fewer islands of skeletal muscle compared to SIS-ECM and a similar fatigue resistance to native muscle. Carbodiimide-crosslinked SIS-ECM and polypropylene mesh were characterized by a chronic inflammatory response and produced little or no measurable tetanic force. The findings of this study show that non-crosslinked xenogeneic SIS scaffolds and autologous tissue are associated with the restoration of functional skeletal muscle with histomorphologic characteristics that resemble native muscle.

Macrophage participation in the degradation and remodeling of extracellular matrix scaffolds.

Biologic scaffolds composed of extracellular matrix (ECM) are widely used to facilitate remodeling and reconstruction of a variety of tissues in both preclinical animal studies and human clinical applications. The mechanisms by which such scaffolds influence the host tissue response are only partially understood, but it is logical that the mononuclear macrophage cell population plays a central role. The present study evaluated the role of macrophages that derive from peripheral blood in the degradation of ECM scaffolds. An established rat body wall reconstruction model was used to evaluate the degradation of carbodiimide (CDI)-crosslinked scaffolds composed of porcine small intestinal submucosa (SIS), noncrosslinked SIS, and autologous body wall. To assess the role of circulating macrophages in the degradation process, the degradation of each scaffold was assessed with and without macrophage depletion caused by administration of clodronate-containing liposomes. Results showed that peripheral blood monocytes are required for the early and rapid degradation of both SIS scaffolds and autologous body wall, and that CDI crosslinked SIS is resistant to macrophage-mediated degradation.

Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component.

Recently, macrophages have been characterized as having an M1 or M2 phenotype based on receptor expression, cytokine and effector molecule production, and function. The effects of macrophage phenotype upon tissue remodeling following the implantation of a biomaterial are largely unknown. The objectives of this study were to determine the effects of a cellular component within an implanted extracellular matrix (ECM) scaffold upon macrophage phenotype, and to determine the relationship between macrophage phenotype and tissue remodeling. Partial-thickness defects in the abdominal wall musculature of Sprague-Dawley rats were repaired with autologous body wall tissue, acellular allogeneic rat body wall ECM, xenogeneic pig urinary bladder tissue, or acellular xenogeneic pig urinary bladder ECM. At 3, 7, 14, and 28 days the host tissue response was characterized using histologic, immunohistochemical, and RT-PCR methods. The acellular test articles were shown to elicit a predominantly M2 type response and resulted in constructive remodeling, while those containing a cellular component, even an autologous cellular component, elicited a predominantly M1 type response and resulted in deposition of dense connective tissue and/or scarring. We conclude that the presence of cellular material within an ECM scaffold modulates the phenotype of the macrophages participating in the host response following implantation, and that the phenotype of the macrophages participating in the host response appears to be related to tissue remodeling outcome.

Oxygen diffusivity of biologic and synthetic scaffold materials for tissue engineering.

Scaffolds for tissue engineering and regenerative medicine applications are commonly manufactured from synthetic materials, intact or isolated components of extracellular matrix (ECM), or a combination of such materials. After surgical implantation, the metabolic requirements of cells that populate the scaffold depend upon adequate gas and nutrient exchange with the surrounding microenvironment. The present study measured the oxygen transfer through three biologic scaffold materials composed of ECM including small intestinal submucosa (SIS), urinary bladder submucosa (UBS), and urinary bladder matrix (UBM), and one synthetic biomaterial, Dacron. The oxygen diffusivity was calculated from Fick's first law of diffusion. Each material permitted measurable oxygen diffusion. The diffusivity of SIS was found to be dependent on the direction of oxygen transfer; the oxygen transfer in the abluminal-to-luminal direction was significantly greater than the luminal-to-abluminal direction. The oxygen diffusivity of UBM and UBS were similar despite the presence of an intact basement membrane on the luminal surface of UBM. Dacron showed oxygen diffusivity values seven times greater than the ECM biomaterials. The current study showed that each material has unique oxygen diffusivity values, and these values may be dependent on the scaffold's ultrastructure.

Macrophage phenotype as a determinant of biologic scaffold remodeling.

Macrophage phenotype can be characterized as proinflammatory (M1) or immunomodulatory and tissue remodeling (M2). The present study used a rat model to determine the macrophage phenotype at the site of implantation of two biologic scaffolds that were derived from porcine small intestinal submucosa (SIS) and that differed mainly according to their method of processing: the Restore device (SIS) and the CuffPatch device (carbodiimide crosslinked form of porcine-derived SIS (CDI-SIS)). An autologous tissue graft was used as a control implant. Immunohistologic methods were used to identify macrophage surface markers CD68 (pan macrophages), CD80 and CCR7 (M1 profile), and CD163 (M2 profile) during the remodeling process. All graft sites were characterized by the dense population of CD68+ mononuclear cells present during the first 4 weeks. The SIS device elicited a predominantly CD163+ response (M2 profile, p < 0.001) and showed constructive remodeling at 16 weeks. The CDI-SIS device showed a predominately CD80+ and CCR7+ response (M1 profile, p < 0.03), and at 16 weeks was characterized by chronic inflammation. The autologous tissue graft showed a predominately CD163+ response (M2) at 1 week, with a dual M1/M2 population (CD80+, CCR7+, and CD163+) by 2 and 4 weeks and moderately well organized connective tissue by 16 weeks. The processing methods used during the manufacturing of a biologic scaffold can have a profound influence upon the macrophage phenotype profile and downstream remodeling events. Routine histologic examination alone is inadequate to determine the phenotype of mononuclear cells that participate in the host response to the scaffold.

Hydrated versus lyophilized forms of porcine extracellular matrix derived from the urinary bladder.

Biologic scaffolds composed of naturally occurring extracellular matrix (ECM) are currently in clinical use for the repair and reconstruction of damaged or missing tissues. The material and structural properties of the ECM scaffold are important determinants of the potential clinical applications and these properties may be affected by manufacturing steps, processing steps, and storage conditions. The present study compared the structural properties of hydrated and lyophilized forms of a biologic scaffold derived from the porcine urinary bladder (urinary bladder matrix or UBM). The structural properties evaluated include: maximum load and elongation, maximum tangential stiffness, energy absorbed, suture retention strength, ball-burst strength, and the hydrostatic permeability index. Other properties that were investigated include changes in the water content, structural morphology, and thickness and the ability to support in vitro growth of NIH 3T3 cells. Lyophilization caused no changes in the structural properties evaluated with the exception of a decrease in maximum elongation. NIH 3T3 cells showed invasion of the scaffold when seeded on the abluminal side of both hydrated and lyophilized UBM, and there were more cells present on lyophilized UBM when compared to hydrated UBM devices after the 7-days culture period. Irreversible changes were observed in the microstructure and ultrastructure of lyophilized UBM devices. We conclude that lyophilization affects the overall in vitro cell growth of NIH 3T3 cells and the ultrastructural morphology of UBM devices, but does not result in significant changes in structural properties.

Extracellular matrix bioscaffolds for orthopaedic applications. A comparative histologic study.

BACKGROUND: Biologic scaffold materials prepared from extracellular matrix are currently available for the surgical repair of damaged or missing musculotendinous tissue. These scaffolds differ in their species and tissue of origin, methods of processing, and methods of terminal sterilization. The purpose of the present study was to evaluate the host-tissue morphologic response to five commercially available extracellular matrix-derived biologic scaffolds used for orthopaedic soft-tissue repair in a rodent model. METHODS: One hundred twenty-six Sprague-Dawley rats were divided into six groups of twenty-one animals each. A defect was created in the musculotendinous tissue of the abdominal wall of each animal and then was repaired with one of five different scaffold materials (GraftJacket, Restore, CuffPatch, TissueMend, Permacol) or with the excised autologous tissue. Three animals from each group were killed at one of seven time-points after surgery (two, four, seven, fourteen, twenty-eight, fifty-six, and 112 days), and the specimens were examined with histologic and morphologic methods. The degree of cellular infiltration, multinucleated giant cell presence, vascularity, and organization of the replacement connective tissue were evaluated with semiquantitative methods. RESULTS: Each device elicited a distinct morphologic response that differed with respect to cellularity (p<0.001), vascularity (p<0.01), the presence of multinucleated giant cells (p<0.01), and organization of the remodeled tissue (p<0.01) at or after the Day 7 time-point. More rapidly degraded devices such as Restore and autologous tissue showed the greatest amount of cellular infiltration, especially at the early time-points. Devices that degraded slowly, such as CuffPatch, TissueMend, and Permacol, were associated with the presence of foreign-body giant cells, chronic inflammation, and/or the accumulation of dense, poorly organized fibrous tissue. CONCLUSIONS: Biologic scaffold materials composed of extracellular matrix elicit distinct host-tissue histologic and morphologic responses, depending on species of origin, tissue of origin, processing methods, and/or method of terminal sterilization.