Effect of the alphaGal epitope on the response to small intestinal submucosa extracellular matrix in a nonhuman primate model.

The Galalpha1,3Galbeta1,4GlcNAc-R (Gal) epitope is a major factor in the hyperacute rejection of pig organ transplants in primates. Biologic scaffold materials used for tissue reconstruction and composed of xenogeneic extracellular matrix (ECM) may contain the Gal epitope. However, the effect of this epitope upon the host response is controversial. The present study investigated the effect of the Gal epitope upon the host response to a porcine-derived ECM in an African Green monkey (Cholrocaebus aethiops) abdominal wall resection model. Histologic methods, serology, complement-dependent cytotoxicity, and gene expression profiling were used to evaluate the host response to allogeneic and both wild-type and Gal-deficient xenogeneic scaffold materials. Although expression of the Gal epitope induced an increase in serum anti-Gal antibodies in recipients, no other differences were noted in the host response between test articles. All ECM scaffolds were well tolerated and showed constructive remodeling during the study period. Recipients of all test articles showed no histologic or humoral evidence of sensitization when a second scaffold was implanted 45 days after the original surgery. The findings of the present study show that the presence of the Gal epitope within a porcine-derived ECM scaffold material elicits a serum antibody response, but no adverse effect upon tissue remodeling.

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.

Chemoattractant activity of degradation products of fetal and adult skin extracellular matrix for keratinocyte progenitor cells.

Biological scaffolds composed of naturally occurring extracellular matrix (ECM) have been utilized as templates for the constructive remodelling of numerous tissues in preclinical studies and human clinical applications. The mechanisms by which ECM induces constructive remodelling are not well understood, but it appears that the degradation products of ECM scaffolds may play key roles in cell recruitment. The objective of the present study was to investigate the effects of age and species of the tissue from which ECM is harvested on the chemoattractant activity of degradation products of ECM for human keratinocyte stem and progenitor cells. Adult human skin ECM, fetal human skin ECM and adult porcine skin ECM were prepared, enzymatically digested, characterized by SDS-PAGE and evaluated for in vitro chemoattractant activity for human keratinocyte progenitor and stem cells (HEKn). Degradation products of human fetal skin ECM showed greater chemoattractant activity than human adult skin ECM degradation products for the HEKn. Degradation products of porcine adult skin ECM showed greater chemoattractant activity than human adult skin ECM. The human fetal skin ECM degradation products showed the strongest chemoattractant activity for the HEKn. The findings of this study support the concept that the mechanism of ECM scaffold remodelling involves the recruitment of lineage-directed progenitor cells by scaffold degradation products, and that both the age and species of the tissue from which the ECM is harvested have an effect upon this chemoattractant potential.

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.

Gene expression by fibroblasts seeded on small intestinal submucosa and subjected to cyclic stretching.

Extracellular matrix scaffolds derived from porcine small intestinal submucosa (SIS-ECM) have been shown to promote the formation of site-specific tissue in a number of preclinical animal studies. However, this constructive remodeling process requires that the scaffold be subjected to a site-specific mechanical environment. The specific quantitative effects of mechanical loading on the gene expression patterns of fibroblasts seeded on SIS-ECM are unknown and yet very important in the tissue remodeling process. The objective of the present study was to evaluate the expression of collagen type I (Col I), collagen type III (Col III), smooth muscle actin (SMA), tenascin-C (TN-C), matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), transforming growth factor-beta1 (TGF-beta1), and transforming growth factor-beta3 (TGF-beta3) by fibroblasts subjected to various magnitudes (0%, 5%, 10%, and 15%) and frequencies (0.1 Hz, 0.3 Hz, and 0.5 Hz) of stretch. A new cyclic-stretching tissue culture (CSTC) system was developed. This system consists of eight independently controlled culture chambers that can be operated in a sterile incubator. Each chamber includes a load cell so that the load in each scaffold can be monitored. It was found that different stretching regimens led to complex and distinctive patterns of gene expression by fibroblasts seeded onto SIS-ECM. In general, the fibroblasts increased expression of Col I up to 5-fold and decreased that of Col III with increased frequency of stretch. In addition, the fibroblasts exhibited a contractile phenotype with increased expression of SMA, TN-C, and TGF-beta1. These findings support the concept that the mechanical environment of a remodeling ECM scaffold may have substantial effects on the behavior of cells within the scaffold and contribute to the site-specific tissue remodeling that has been observed in in vivo studies.

Degradation and remodeling of small intestinal submucosa in canine Achilles tendon repair.

BACKGROUND: Extracellular matrix derived from porcine small intestinal submucosa is used for the repair of musculotendinous tissues. Preclinical evaluation and clinical use have suggested that small intestinal submucosa extracellular matrix degrades rapidly after implantation and can be replaced by host tissue that is functionally and histologically similar to the normal tissue. METHODS: The present study analyzed the temporal degradation of a ten-layer multilaminate device of small intestinal submucosa extracellular matrix used for the repair of canine Achilles tendon and examined the corresponding histological appearance of the remodeled tissue during the course of scaffold degradation. Devices were fabricated from small intestinal submucosa extracellular matrix labeled with 14C. The amount of 14C remaining in the remodeled graft was measured by liquid scintillation counting at three, seven, fourteen, twenty-eight, sixty, and ninety days after surgery. Blood, urine, feces, and other parenchymal tissues were also harvested to determine the fate of scaffold degradation products. Tissue specimens were prepared for routine histological analysis to examine the morphology of the remodeled graft at each time-point. RESULTS: The small intestinal submucosa extracellular matrix graft degraded rapidly, with approximately 60% of the mass lost by one month after surgery, and the graft was completely resorbed by three months after surgery. The graft supported rapid cellular infiltration and host tissue ingrowth. By ninety days after surgery, the remodeled small intestinal submucosa extracellular matrix consisted of a dense collagenous tissue with organization, cellularity, and vascularity similar to that of normal tendon. CONCLUSIONS: Small intestinal submucosa extracellular matrix is rapidly degraded after implantation for the repair of a musculotendinous tissue in this canine Achilles tendon repair model and is replaced by the deposition and organization of host tissue that is histologically similar to that of normal tissue.

A quantitative method for evaluating the degradation of biologic scaffold materials.

Scaffolds derived from naturally occurring extracellular matrix (ECM) have found extensive use in the fields of tissue engineering and regenerative medicine. Many of these scaffolds are designed to degrade rapidly as they are replaced by new host tissue. Other scaffolds are chemically crosslinked to slow the rate of degradation or add strength to the scaffold. Commercially available ECM scaffolds have considerable variability with regards to tissue origin and methods of processing, and little is known about their rate of degradation and the fate of their degradation products. A novel method is described herein to integrally label ECM with a radioactive isotope ((14)C). It was found that a number of tissues are efficiently labeled, including heart, liver, trachea, pancreas, small intestine, and urinary bladder tissue. Of the tissues analyzed, only spleen was not found to contain detectable levels of (14)C. The technique is extremely sensitive, accurate, and safe, but requires access to accelerator mass spectrometry, and is expensive and time consuming. This model represents the first described quantitative method to determine the rate of degradation for an ECM scaffold and to track the fate of the degradation products.

Effects of exogenous estrogen on uterine leukocyte recruitment.

OBJECTIVE: To determine the role of estrogen in leukocyte recruitment to the human endometrium. DESIGN: Prospective, controlled in vivo study. SETTING: Academic research laboratory. PATIENT(S): Ten patients presenting for donor oocytes. INTERVENTION(S): Endometrial biopsies for the evaluation of leukocyte populations were collected from perimenopausal women in two consecutive regulated cycles who were given two different regimens of estrogen with identical progesterone treatment. MAIN OUTCOME MEASURE(S): Immunohistochemical identification of endometrial leukocyte populations and relative levels of expression of three chemokine genes. RESULT(S): The total uterine leukocyte population increased significantly when the women received oral estrogen, which resulted in higher serum estrogen levels. This rise in leukocytes was due to a significant increase in both the uterine natural killer cells and the macrophage populations. T-cell numbers did not change relative to circulating estrogen levels. The relative abundance of mRNA from three chemokines was also determined. No changes were found in the expression of M-CSF or MCP-1. Interleukin 8 decreased in glands relative to estrogen levels. CONCLUSION(S): These data demonstrate that changes in circulating levels of estrogen can regulate the recruitment of bone marrow-derived cells to the uterine endometrium; however, the mechanism whereby that occurs remains elusive.