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.

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 L 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.

An abundant quantity of mesenchymal stem cells can be isolated from adipose tissue during standard cosmetic procedures. Alongside adipocytes, the stromal vascular fraction (SVF) includes adipose stem/progenitor cells (ASCs), mature and immature vascular endothelial cells, pericytes, fibroblasts, and hematopoietic cells. ASCs are highly proliferative and differentiate into adipocytes, chondrocytes, osteoblasts, and myocytes. ASCs have immunomodulatory properties, and, therefore, can be administered as an autogenic or allogenic cell product. SVF can be isolated using automated devices or manually. After isolation from adipose tissue, SVF can be admixed into lipoaspirate for cell-enhanced fat grafting (cell-assisted lipotransfer), locally injected into areas of injury, or given intravenously. In the US, the FDA classifies ASCs into one of two categories: human cells and tissue products, or as a “biologic drug.” There are over 140 ongoing clinical trials globally that include ASCs in therapies for various diseases or injuries.

Abstract Introduction: Breast surgery often results in physical deformities that significantly diminish patient's quality of life. A natural and aesthetically pleasing breast reconstruction can be achieved through autologous fat grafting and the success of this technique has largely been attributed to the regenerative properties of adipose-derived stem cells (ASCs) within fat graft. However, in the setting of resected breast cancer, the growth stimulating and angiogenic effects of ASCs pose a risk of increasing local recurrence rates. Although the safety of autologous fat grafting in the breast cancer population has yet to be determined, potential recurrence risk may be minimized through incorporating tumor-suppressing elements in the graft. This study aimed to determine if doxorubicin and paclitaxel could be used to inhibit breast cancer cells while maintaining the viability and functionality of ASCs in vitro. Materials and methods: Human ASCs were isolated from non-diabetic female patients between 35 and 60 years of age (n = 3). BT-474 and MDA-MB-231 were obtained from ATCC. ASCs, ASCs undergoing adipogenic differentiation, and breast cancer cells were each exposed to a range of doxorubicin-HCl (0, 10, 30, 100, 300, 1000, 3000, or 10000nM) or paclitaxel (0, 0.1, 0.3, 1, 3, 10, 30, 100, or 300nM) concentrations. Proliferation, viability, and differentiation capacity were assessed with commercially available CyQuant, MTT, and AdipoRed assay kits, respectively. Results: Dose-dependent inhibition was observed for doxorubicin in ASCs and both breast cancer cell lines. The IC50 of doxorubicin on ASCs, BT-474, and MDA-MB-231 were 901.3, 656.5, and 333 nM, respectively. Dose-dependent inhibition for paclitaxel was only observed in BT-474 and MDA-MB-231 cells, with IC50 values of 1.809 and 3.477 nM, respectively. ASCs maintained greater than 80% viability over the range of paclitaxel concentrations tested and consequently and IC50 value could not be determined. ASCs differentiation into mature adipocytes was not inhibited by doxorubicin exposure. Discussion: In vitro cytotoxicity studies demonstrated greater doxorubicin and paclitaxel sensitivity in BT-474 and MDA-MB-231 than in ASCs. Furthermore, the presence of doxorubicin did not inhibit ASC differentiation into mature adipocytes. These findings suggest that incorporating chemotherapeutic drugs in fat grafts for breast reconstruction following primary breast cancer surgery may be a viable option for decreasing the risk of cancer recurrence. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-03.

The remodeling response to extracellular matrix (ECM) scaffold materials such as porcine small intestinal submucosa (SIS) is characterized by intense mononuclear cell infiltration during the first 4 weeks post-implantation. Persistence of macrophages in wounds is typically diagnosed as chronic inflammation with downstream formation of scar tissue and/or foreign body reaction, but ECM scaffolds remodel into organized site-specific tissue. Macrophages can express either proinflammatory (M1) or immunomodulatory and tissue remodeling (M2) phenotypes. Processing methods used during the manufacturing of ECM scaffolds can influence macrophage phenotype and downstream remodeling outcome.In the first study, human monocyte-derived macrophages were cultured on SIS and carbodiimide (CDI) crosslinked SIS in 20% and 6% oxygen concentrations. Macrophage phenotype was evaluated by expression of M1 (CXCL10 and CCR7) and M2 (ARG-1, CCL13, CCL18, and MRC-1) gene markers, and secretion of CXCL10, CCL13, CCL18, and MMP9. Macrophages cultured on SIS expressed an M2 profile, while macrophages cultured on CDI-SIS expressed a mixed M1/M2 profile. No consistent patterns were observed when comparing oxygen concentrations.The second study used radioactive 14C-labeled scaffolds to measure ECM scaffold degradation in a rodent model of musculoskeletal reconstruction with and without the depletion of macrophages. Tissues were characterized by expression of M1 (iNOS and IFN-a) and M2 (ARG-1 and IL-10) gene markers, and cell surface markers CD68 (pan-macrophage), CCR7 (M1), and CD163 (M2). Results showed that macrophages are required for early and rapid degradation of SIS scaffolds, and that CDI-SIS is resistant to macrophage-mediated degradation. Furthermore, depletion of macrophages resulted in an attenuated inflammatory response and slowed the rate of scaffold degradation.The third study determined the contractile response and histomorphologic appearance of tissue repaired with SIS, CDI-SIS, or autologous tissue at 26 weeks after implantation. Contractile properties and fatigue resistance of remodeled tissue and of contralateral native tissue were assessed using an in-situ methodology. Muscle fiber-type distribution, blood vessel density and distribution, and innervation were determined. The tissue repaired with SIS showed complete replacement by tissue that histologically and functionally resembled native muscle. CDI-SIS was characterized by chronic inflammatory response and produced little to no measurable tetanic force output.

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.

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.

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.

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 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.

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. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 87A: 862-872, 2008

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. Clinical Relevance: The temporal sequence of remodeling events of extracellular matrix devices, including the rapidity of scaffold degradation and the extent of new-tissue deposition by the host, may be predictive of the clinical course and may determine the optimal rehabilitation protocol and functional outcome of the procedure.

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.Clinical Relevance: The temporal sequence of remodeling events of extracellular matrix devices, including the rapidity of scaffold degradation and the extent of new-tissue deposition by the host, may be predictive of the clinical course and may determine the optimal rehabilitation protocol and functional outcome of the procedure.