Matrix-bound nanovesicle-associated IL-33 supports functional recovery after skeletal muscle injury by initiating a pro-regenerative macrophage phenotypic transition.

Injuries to skeletal muscle are among the most common injuries in civilian and military populations, accounting for nearly 60% of extremity injuries. The standard of care for severe extremity injury has been focused upon limb salvage procedures and the utilization of tissue grafts or orthotics in conjunction with rehabilitation to avoid amputation. Nonetheless, many patients have persistent strength and functional deficits that permanently impact their quality of life. Preclinical and clinical studies have shown that partial restoration of functional skeletal muscle tissue following injury can be achieved by the implantation of a biologic scaffold composed of extracellular matrix (ECM). These favorable outcomes are mediated, at least in part, through local immunomodulation. The mechanisms underlying this immunomodulatory effect, however, are poorly understood. The present study investigates a potential mechanistic driver of the immunomodulatory effects; specifically, the effect of selected ECM components upon inflammation resolution and repair. Results show that the host response to skeletal muscle injury is profoundly altered and functional recovery decreased in il33-/- mice compared to age- and sex-matched wildtype counterparts by 14 days post-injury. Results also show that IL-33, contained within matrix-bound nanovesicles (MBV), supports skeletal muscle regeneration by regulating local macrophage activation toward a pro-remodeling phenotype via canonical and non-canonical pathways to improve functional recovery from injury compared to untreated il33-/- counterparts. Taken together, these data suggest that MBV and their associated IL-33 cargo represent a novel homeostatic signaling mechanism that contributes to skeletal muscle repair.

Menisco-fibular ligament - an overview: cadaveric dissection, clinical and magnetic resonance imaging diagnosis, arthroscopic visualisation and treatment.

BACKGROUND: Injury to the menisco-fibular ligament (MFiL) is not commonly recognised. The anatomy of the lateral meniscus is complex and structure-function relationships are only partly understood. The purpose of the present study was to evaluate the MFiL, an anatomic structure rarely discussed that stabilises the lateral meniscus at the level of the hiatus popliteus and may have a crucial role in pathology of lateral meniscus injury. MATERIALS AND METHODS: The MFiL was dissected from its attachment at the lateral meniscus to its insertion on fibular head in 12 human normal cadaver knees. The dimensions were determined and its anatomic position visualised throughout a 90° range of motion. Findings were documented on digital photographs and on video. Results were compared against the magnetic resonance imaging (MRI) appearance of the injured MFiL in 20 patients. Concomitant knee injuries in those patients were also analysed to determine the most frequent pattern of injuries. RESULTS: The normal MFiL showed an inverted trapezoid-shape with a mean width proximally of 13 mm, mean width distally of 8.5 mm and a mean length of 18.4 mm. MRI visualisation of the ligament was possible even in regular sequences; however, additional radial plane sequences were also used. Arthroscopic visualisation and manipulation was optimal when the camera was inserted into the postero-lateral gutter with full knee extension. CONCLUSIONS: The MFiL stabilises the postero-lateral knee in concert with the menisco-femoral ligaments. Injury to the MFiL can be a cause of chronic postero-lateral pain syndrome with associated instability. Further anatomical and biomechanical studies are needed in order to fully evaluate its importance.

Mechanical strength vs. degradation of a biologically-derived surgical mesh over time in a rodent full thickness abdominal wall defect.

The use of synthetic surgical mesh materials has been shown to decrease the incidence of hernia recurrence, but can be associated with undesirable effects such as infection, chronic discomfort, and adhesion to viscera. Surgical meshes composed of extracellular matrix (i.e., biologically-derived mesh) are an alternative to synthetic meshes and can reduce some of these undesirable effects but are less frequently used due to greater cost and perceived inadequate strength as the mesh material degrades and is replaced by host tissue. The present study assessed the temporal association between mechanical properties and degradation of biologic mesh composed of urinary bladder matrix (UBM) in a rodent model of full thickness abdominal wall defect. Mesh degradation was evaluated for non-chemically crosslinked scaffolds with the use of (14)C-radiolabeled UBM. UBM biologic mesh was 50% degraded by 26 days and was completely degraded by 90 days. The mechanical properties of the UBM biologic mesh showed a rapid initial decrease in strength and modulus that was not proportionately associated with its degradation as measured by (14)C. The loss of strength and modulus was followed by a gradual increase in these values that was associated with the deposition of new, host derived connective tissue. The strength and modulus values were comparable to or greater than those of the native abdominal wall at all time points.

The effect of detergents on the basement membrane complex of a biologic scaffold material.

The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to 3% Triton-X 100, 8mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24h. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA, glycosaminoglycans (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin ß-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8mM CHAPS and 1% SDS. Both 8mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC were able to infiltrate the damaged sub-basement membrane tissue, showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold.

Fractionation of an ECM hydrogel into structural and soluble components reveals distinctive roles in regulating macrophage behavior.

Extracellular matrix (ECM) derived from mammalian tissues has been utilized to repair damaged or missing tissue and improve healing outcomes. More recently, processing of ECM into hydrogels has expanded the use of these materials to include platforms for 3-dimensional cell culture as well as injectable therapeutics that can be delivered by minimally invasive techniques and fill irregularly shaped cavities. At the cellular level, ECM hydrogels initiate a multifaceted host response that includes recruitment of endogenous stem/progenitor cells, regional angiogenesis, and modulation of the innate immune response. Unfortunately, little is known about the components of the hydrogel that drive these responses. We hypothesized that different components of ECM hydrogels could play distinctive roles in stem cell and macrophage behavior. Utilizing a well-characterized ECM hydrogel derived from urinary bladder matrix (UBM), we separated the soluble and structural components of UBM hydrogel and characterized their biological activity. Perivascular stem cells migrated toward and reduced their proliferation in response to both structural and soluble components of UBM hydrogel. Both components also altered macrophage behavior but with different fingerprints. Soluble components increased phagocytosis with an IL-1RA(high), TNFα(low), IL-1ß(low), uPA(low) secretion profile. Structural components decreased phagocytosis with a PGE2(high), PGF2α(high), TNFα(low), IL-1ß(low), uPA(low), MMP2(low), MMP9(low), secretion profile. The biologic activity of the soluble components was mediated by Notch and PI3K/Akt signaling, while the biologic activity of the structural components was mediated by integrins and MEK/ERK signaling. Collectively, these findings demonstrate that soluble and structural components of ECM hydrogels contribute to the host response but through different mechanisms.

Hydrogels derived from demineralized and decellularized bone extracellular matrix.

The extracellular matrix (ECM) of mammalian tissues has been isolated, decellularized and utilized as a scaffold to facilitate the repair and reconstruction of numerous tissues. Recent studies have suggested that superior function and complex tissue formation occurred when ECM scaffolds were derived from site-specific homologous tissues compared with heterologous tissues. The objectives of the present study were to apply a stringent decellularization process to demineralized bone matrix (DBM), prepared from bovine bone, and to characterize the structure and composition of the resulting ECM materials and DBM itself. Additionally, we sought to produce a soluble form of DBM and ECM which could be induced to form a hydrogel. Current clinical delivery of DBM particles for treatment of bone defects requires incorporation of the particles within a carrier liquid. Differences in osteogenic activity, inflammation and nephrotoxicity have been reported with various carrier liquids. The use of hydrogel forms of DBM or ECM may reduce the need for carrier liquids. DBM and ECM hydrogels exhibited sigmoidal gelation kinetics consistent with a nucleation and growth mechanism, with ECM hydrogels characterized by lower storage moduli than the DBM hydrogels. Enhanced proliferation of mouse primary calvarial cells was achieved on ECM hydrogels, compared with collagen type I and DBM hydrogels. These results show that DBM and ECM hydrogels have distinct structural, mechanical and biological properties and have the potential for clinical delivery without the need for carrier liquids.

Neurorestorative effect of urinary bladder matrix-mediated neural stem cell transplantation following traumatic brain injury in rats.

Traumatic brain injury (TBI) is a leading cause of cell death and disability among young adults and lacks a successful therapeutic strategy. The multiphasic injuries of TBI severely limit the success of conventional pharmacological approaches. Recent successes with transplantation of stem cells in bioactive scaffolds in other injury paradigms provide new hope for the treatment of TBI. In this study, we transplanted neural stem cells (0.5x10(5) cells/µl) cultured in a bioactive scaffold derived from porcine urinary bladder matrix (UBM; 4 injection sites, 2.5µl each) into the rat brain following controlled cortical impact (CCI, velocity, 4.0 m/sec; duration, 0.5 sec; depth, 3.2mm). We evaluated the effectiveness of this strategy to combat the loss of motor, memory and cognitive faculties. Before transplantation, compatibility experiments showed that UBM was able to support extended proliferation and differentiation of neural stem cells. Together with its reported anti-inflammatory properties and rapid degradation characteristics in vivo, UBM emerged to be an ideal scaffold. The transplants reduced neuron/tissue loss and white matter injury, and also significantly ameliorated motor, memory, and cognitive impairments. Furthermore, exposure to UBM alone was sufficient to decrease the loss of sensorimotor skills from TBI (examined 3-28 days post-CCI). However, only UBMs that contained proliferating neural stem cells helped attenuate memory and cognitive impairments (examined 26-28 days post-CCI). In summary, these results demonstrate the therapeutic efficacy of stem cells in bioactive scaffolds against TBI and show promise for translation into future clinical use.

Damage associated molecular patterns within xenogeneic biologic scaffolds and their effects on host remodeling.

The immune response is an important determinant of the downstream remodeling of xenogeneic biologic scaffolds in vivo. Pro-inflammatory responses have been correlated with encapsulation and a foreign body reaction, while anti-inflammatory reactions are associated with constructive remodeling. However, the bioactive and bioinductive molecules within the extracellular matrix (ECM) that induce this polarization are unclear, although it is likely that cellular remnants such as damage associated molecular patterns (DAMPs) retained within the scaffold may play a role. The present study investigated the immunomodulatory effects of common ECM scaffolds. Results showed that tissue source, decellularization method and chemical crosslinking modifications affect the presence of the well characterized DAMP - HMGB1. In addition, these factors were correlated with differences in cell proliferation, death, secretion of the chemokines CCL2 and CCL4, and up regulation of the pro-inflammatory signaling receptor toll-like receptor 4 (TLR4). Inhibition of HMGB1 with glycyrrhizin increased the pro-inflammatory response, increasing cell death and up regulating chemokine and TLR4 mRNA expression. The present study suggests the importance of HMGB1 and other DAMPS as bioinductive molecules within the ECM scaffold. Identification and evaluation of other ECM bioactive molecules will be an area of future interest for new biomaterial development.

In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering.

The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL) for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM) seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL) and cells in an intact ACL (iACL), particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA). Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.

Thrombolysis vs. bleeding from hemostatic sites by a prourokinase mutant compared with tissue plasminogen activator.

BACKGROUND: A single site mutant (M5) of prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. METHODS AND RESULTS: After completing dose-finding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg(-1)) or tissue plasminogen activator (t-PA) (1.4 mg kg(-1)) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean approximately 40 mL) than with M5 (mean approximately 4 mL) (P = 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. CONCLUSIONS: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.

Marrow-derived cells populate scaffolds composed of xenogeneic extracellular matrix.

INTRODUCTION: The source of cells that participate in wound repair directly affects outcome. The extracellular matrix (ECM) and other acellular biomaterials have been used as therapeutic scaffolds for cell attachment and proliferation and as templates for tissue repair. The ECM consists of structural and functional proteins that influence cell attachment, gene expression patterns, and the differentiation of cells. OBJECTIVE: The objective of this study was to determine if the composition of acellular matrix scaffolds affects the recruitment of bone marrow-derived cellular elements that populate the scaffolds in vivo. METHODS: Scaffolds composed of porcine tissue ECM, purified Type I collagen, poly(L)lactic coglycolic acid (PLGA), or a mixture of porcine ECM and PLGA were implanted into subcutaneous pouches on the dorsum of mice. The origin of cells that populated the matrices was determined by first performing bone marrow transplantation to convert the marrow of glucose phosphate isomerase 1b (Gpi-1(b)) mice to cells expressing glucose phosphate isomerase 1a (Gpi-1(a)). RESULTS: A significant increase in Gpi-1(a) expressing cells was present in sites implanted with the porcine ECM compared to sites implanted with either Type I collagen or PLGA. Use of recipient mice transplanted with marrow cells that expressed beta-galactosidase confirmed that the majority of cells that populated and remodeled the naturally occurring porcine ECM were marrow derived. Addition of porcine ECM to the PLGA scaffold caused a significant increase in the number of marrow-derived cells that became part of the remodeled implant site. CONCLUSION: The composition of bioscaffolds affects the cellular recruitment pattern during tissue repair. ECM scaffolds facilitate the recruitment of marrow-derived cells into sites of remodeling.

In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair.

The rate of in vivo degradation was determined for a naturally occurring biomaterial derived from the extracellular matrix of the small intestinal submucosa (SIS). The SIS was labeled by giving weekly intravenous injections of 10 microCi of 14C-proline to piglets from 3 weeks of age until the time of sacrifice at 26 weeks. The resultant SIS prepared from these pigs contained approximately 10(3) fold more 14C than unlabeled tissues. The labeled SIS was used to repair experimental defects in the urinary bladder of 10 dogs. The animals were sacrificed at post-operative times ranging from 3 days to 1 year and the remodeled urinary bladder tissue was harvested for evaluation of 14C by a combination of liquid scintillation counting and accelerator mass spectrometry. The remodeled tissue contained less than 10% of the 14C (disintegrations per minute/gram tissue wet weight) at 3 months post-surgery compared to the SIS biomaterial that was originally implanted. The SIS scaffold was replaced by host tissue that resembled normal bladder both in structure and function. After implantation, 14C was detected in highest concentrations in the blood and the urine. The SIS bioscaffold provides a temporary scaffold for tissue remodeling with rapid host tissue remodeling, degradation, and elimination via the urine when used as a urinary bladder repair device.

Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response.

BACKGROUND: Porcine small intestinal submucosa (SIS) is an acellular, naturally derived extracellular matrix (ECM) that has been used for tissue remodeling and repair in numerous xenotransplantations. Although a vigorous immune response to xenogeneic extracellular matrix biomaterials is expected, to date there has been evidence for only normal tissue regeneration without any accompanying rejection. The purpose of this study was to determine the reason for a lack of rejection. METHODS: Mice were implanted s.c. with xenogeneic tissue, syngeneic tissue, or SIS, and the graft site analyzed histologically for rejection or acceptance. Additionally, graft site cytokine levels were determined by reverse transcriptase polymerase chain reaction and SIS-specific serum antibody isotype levels were determined by ELISA. RESULTS: Xenogeneically implanted mice showed an acute inflammatory response followed by chronic inflammation and ultimately graft necrosis, consistent with rejection. Syngeneically or SIS implanted mice, however, showed an acute inflammatory response that diminished such that the graft ultimately became indistinguishable from native tissue, observations that are consistent with graft acceptance. Graft site cytokine analysis showed an increase in interleukin-4 and an absence of interferon-gamma. In addition, mice implanted with SIS produced a SIS-specific antibody response that was restricted to the IgG1 isotype. Reimplantation of SIS into mice led to a secondary anti-SIS antibody response that was still restricted to IgG1. Similar results were observed with porcine submucosa derived from urinary bladder. To determine if the observed immune responses were T cell dependent, T cell KO mice were implanted with SIS. These mice expressed neither interleukin-4 at the implant site nor anti-SIS-specific serum antibodies but they did accept the SIS graft. CONCLUSIONS: Porcine extracellular matrix elicits an immune response that is predominately Th2-like, consistent with a remodeling reaction rather than rejection.

Small bowel tissue engineering using small intestinal submucosa as a scaffold.

BACKGROUND: Small intestinal submucosa (SIS) is an extracellular matrix used in tissue engineering studies to create de novo abdominal wall, urinary bladder, tendons, blood vessels, and dura mater. The purpose of this study is to evaluate the feasibility of using SIS as a scaffold for small bowel regeneration in an in situ xenograft model. MATERIALS AND METHODS: Twenty-three dogs had a partial defect created on the small bowel wall which was repaired with a SIS patch. Four dogs underwent small bowel resection with placement of an interposed tube of SIS. The animals were followed 2 weeks to 1 year. RESULTS: Three of the 23 dogs with SIS placed as a patch died shortly after surgery due to leakage from the site. The other 20 dogs survived up to time of elective necropsy with no evidence of intestinal dysfunction. At necropsy, the bowel circumference in the patched area had no stenosis. Histological evaluation showed the presence of a mucosal epithelial layer, varying amount of smooth muscle, sheets of collagen, and a serosal covering. Architecturally, the layers were not well organized in the submucosal region. An abundance of inflammatory cells was present in the early postoperative period but receded with time. All 4 dogs with a tubular segment of SIS interposed had significant problems. One had partial obstruction at 1 month, and 3 died in the early postoperative period due to leakage. CONCLUSIONS: This preliminary study suggests that SIS patches can be used for small bowel regeneration. Tubular segmental replacement is not feasible at this time.

Vascular endothelial growth factor in porcine-derived extracellular matrix.

An extracellular matrix (ECM) derived from the submucosa of the porcine small intestine (SIS) has been shown to induce angiogenesis and host tissue remodeling when used as a xenogeneic bioscaffold in animal models of wound repair. In the present study, we compared the in vitro effects of SIS ECM extracts to several purified angiogenic growth factors on human dermal microvascular endothelial cell (HMEC) growth patterns. The SIS ECM was shown to induce tube formation from HMEC in a three-dimensional fibrin-based angiogenesis assay in a manner similar to that caused by the addition of vascular endothelial growth factor (VEGF). This tube formation was blocked in the presence of anti-VEGF neutralizing antibody. Western blots and ELISA procedures showed that the SIS ECM contains as much as 0.77 ng VEGF/g SIS. The closely related endothelial cell mitogen, platelet-derived growth factor (PDGF), was not detectable in the SIS extracts. We conclude that VEGF is present in the SIS extracellular matrix. The role of VEGF in SIS-induced wound repair remains unknown, but its presence in the ECM makes it a possible contributor to the angiogenic effect of SIS when this ECM is used as a tissue repair scaffold in animal models of wound repair.

Porcine small intestinal submucosa (SIS): a bioscaffold supporting in vitro primary human epidermal cell differentiation and synthesis of basement membrane proteins.

The growth pattern of human epidermal cells, fibroblasts or Swiss mouse 3T3/J2 fibroblasts cultured upon the extracellular matrix (ECM) derived from small intestinal submucosa (SIS) was evaluated. The cell/SIS composites were grown submerged, then maintained in air/liquid interface for 2, 7, 10 or 14 days. The presence of differentiation-related keratins 10, 14 and 16, FN, laminin, collagen type VII and collagen type IV was determined by immunohistochemical methods in SIS alone and in the SIS/cell composite. Only FN could be detected in SIS alone. SIS supported the formation of an epithelial structure with suprabasal expression of K16 and regional suprabasal expression of K10. The epidermal cells were K14 positive and tended to 'invade' the SIS to various degrees. Following the growth of epidermal cells and fibroblasts on the SIS substratum, immunolabeling of FN, laminin, collagen type VII and collagen type IV was observed in a cell-associated pattern. The fibroblasts commonly invaded the SIS, when co-cultivated with epidermal cells on the opposite side of the SIS. The ability of SIS to support epidermal cell/fibroblast attachment, migration and/or proliferation and differentiation with deposition of basement membrane (BM) components indicates that the composite model may be useful for studying cell-matrix interactions and for investigation as a dermal substitute.

Galalpha(1,3)Gal epitope in porcine small intestinal submucosa.

Small intestinal submucosa (SIS) is a naturally occurring, acellular biomaterial derived from porcine jejunum, which promotes constructive tissue remodeling when applied as a xenogeneic graft material. Galactosyl-alpha(1,3)galactose (Gal) is a cell-associated epitope responsible for hyperacute rejection of porcine whole-organ xenografts in primates. Because SIS is harvested from porcine tissue, it may contain the Gal epitope. The goals of this study were to determine if Gal is present in SIS and, if it is present, to determine if human serum complement can be activated in vitro following exposure to porcine-derived SIS. SIS was probed for Gal by immunohistochemical methods and by lectin-peroxidase staining. SIS stained strongly positive with human serum, which contains naturally occurring antibodies to Gal, followed by anti-immunoglobulin G (IgG) or anti-IgM peroxidase conjugate. Blocking with the lectin I-B(4), which is specific for the Gal epitope, decreased the intensity of staining. Exposure of SIS to alpha-galactosidase reduced staining to negligible amounts. The Gal epitope is distributed transmurally throughout the SIS material. Subtyping of the immunoglobulins that bind to SIS showed that IgG(2) is the major immunoglobulin of human plasma that binds to SIS. SIS did not activate complement in vitro as measured by radioimmunoassay for C3a.

Enhanced bone regeneration using porcine small intestinal submucosa.

Small intestinal submucosa (SIS) is an easily produced material that has been used experimentally for tissue engineering. To evaluate the ability of SIS to facilitate bone growth within a long-bone defect, a segment of the radius was surgically removed in adult, female Sprague-Dawley rats. The defect was either left unfilled or implanted with SIS, demineralized cortical bone (DMCB), or ovalbumin. The defect was evaluated radiographically and histologically after 3, 6, 12, and 24 weeks. Tissue remodeling within the defect was evident by week 3 in SIS- and DMCB-treated rats. Filling was characterized initially by infiltration of mononuclear cells and extracellular material in SIS-implanted rats and multifocal remodeling bone particles and cartilage formation in DMCB-implanted rats. Cartilage was observed as early as 3 weeks and bone as early as 6 weeks in SIS-implanted rats. Filling of the defect arose from multiple foci in DMCB-implanted rats, but was contiguous with and parallel to the ulnar shaft in SIS-implanted rats, suggesting that defect repair by SIS may be conductive rather than inductive. Rats in which the defect was left unfilled demonstrated slow but progressive filling of the defect, characterized by mononuclear cell infiltrates and fibrous extracellular material. In summary, SIS facilitated rapid filling of a long-bone defect. These results suggest that SIS may be useful as a bone repair material.