A high-potential therapy for repairing the heart post-myocardial infarction is the implantation of tissueengineered myocardium. While several groups have developed constructs that mimic the aligned structure of the native myocardium, to date no one has investigated the particular functional benefits conferred by alignment. In this study we created myocardial constructs in both aligned and isotropic configurations by entrapping neonatal rat cardiac cells in fibrin gel. Constructs were cultured statically for 2 weeks, and then characterized. Histological staining showed spread cells that express typical cardiac cell markers in both configurations. Isotropic constructs had higher final cell and collagen densities, but lower passive mechanical properties than aligned constructs. Twitch force associated with electrical pacing, however, was 181% higher in aligned constructs, and this improvement was greater than what would be expected from merely aligning the cells in the isotropic constructs in the force measurement direction. Our hypothesis was that this was due to improved gap junction formation=function facilitated by cell alignment, and further analyses of the twitch force data, as well as Western blot results of connexin 43 expression and phosphorylation state, support this hypothesis. Regardless of the specific mechanism, the results presented in this study underscore the importance of recapitulating the anisotropy of the native tissue in engineered myocardium.

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight component of the fibrillin-rich microfibril. Gene-targeted inactivation of MAGP-1 reveals a complex phenotype that includes increased body weight and size due to excess body fat, an altered wound healing response in bone and skin, and a bleeding diathesis. Elastic tissues rich in MAGP-1-containing microfibrils develop normally and show normal function. The penetrance of MAGP-1-null phenotypes is highly variable and mouse strain-dependent, suggesting the influence of modifier genes. MAGP-1 was found to bind active transforming growth factor-␤ (TGF-␤) and BMP-7 with high affinity, suggesting that it may be an important modulator of microfibrilmediated growth factor signaling. Many of the phenotypic traits observed in MAGP-1-deficient mice are consistent with loss of TGF-␤ function and are generally opposite those associated with mutations in fibrillin-1 that result in enhanced TGF-␤ signaling. Increased body size and fat deposition in MAGP-1-mutant animals are particularly intriguing given the localization of obesity traits in humans to the region on chromosome 1 containing the MAGP-1 gene.Microfibrils are important contributors to the structural integrity of tissues and participate in the assembly of elastic fibers during development. They also serve to bind and sequester growth factors in the extracellular matrix (ECM) 3 (1, 2) and can directly signal cells through sequence motifs that interact with integrins and other cell-surface receptors (3-6). The major structural components of these microfibrils are the fibrillins, large glycoproteins rich in calcium binding epidermal growth factor-like domains (7), and the MAGPs, small, cysteine-rich proteins of unknown function. Other proteins can be localized to microfibrils, but it is not clear whether they are integral or associated proteins (8).MAGP-1, a ϳ20-kDa glycoprotein, is synthesized by most matrix-producing cells (9). The N-terminal half of the molecule contains sites for tyrosine sulfation and transglutaminase crosslinking as well as all of the tri-and tetrasaccharide O-linked sugars (10). The first 20 amino acids of the protein are enriched in acidic residues that, together with the sulfotyrosines, create a region of high negative charge capable of binding cationic proteins. The C-terminal half contains all of the molecule's thirteen cysteine residues and encodes a 54-amino acid sequence that defines a matrix-binding domain that targets MAGP-1 to the ECM. MAGP-1 binds to tropoelastin and type VI collagen (11, 12) and interacts with other molecules with defined structural roles in the ECM such as fibrillin-1 and -2 (13, 14), decorin (15), and biglycan (16). It does not, however, bind to the interstitial collagens I, III, or V (12). MAGP-1 also interacts with and facilitates the shedding of Notch1 (17), but there is no evidence for interaction with integrins.MAGP-2 is the other member of the MAGP family and, like MAGP-1, is covalently associated with fibrillin-containing microfibrils (...

Completely biological tissue replacements can be fabricated by entrapping cells in a molded fibrin gel. Over time, the fibrin is degraded and replaced with cell-produced extracellular matrix. However, the relationship between fibrin degradation and matrix deposition has not been elucidated. We developed techniques to quantify fibrin degradation products (FDP) and examine plasmin activity in the conditioned medium from fibrin-based constructs. Fibrin-based tissue constructs fabricated with vascular smooth muscle cells (vSMC) were cultured for 5 weeks in the presence of varied concentrations of the fibrinolysis inhibitor -aminocaproic acid and cellularity, and deposited collagen and elastin were measured weekly. These data revealed that increasing concentrations of -aminocaproic acid led to delayed and diminished FDP production, lower vSMC proliferation, and decreased collagen and elastin deposition. FDP were shown to have a direct biological effect on vSMC cultures and vSMC within the fibrin-based constructs. Supplementing construct cultures with 250 or 500μg/mL FDP led to 30% higher collagen deposition than the untreated controls. FDP concentrations as high as 250μg/mL were estimated to exist within the constructs, indicating that FDP generation during remodeling of the fibrin-based constructs exerted direct biological activity. These results help explain many of the positive outcomes reported with fibrin-based tissue constructs in the literature, as well as demonstrate the importance of regulating plasmin activity during their fabrication.

No abstract

MAGP1 is an extracellular matrix protein that, in vertebrates, is a ubiquitous component of fibrillin-rich microfibrils. We previously reported that aged MAGP1-deficient mice (MAGP1⌬) develop lesions that are the consequence of spontaneous bone fracture. We now present a more defined bone phenotype found in MAGP1⌬mice. A longitudinal DEXA study demonstrated age-associated osteopenia in MAGP1⌬ animals and CT confirmed reduced bone mineral density in the trabecular and cortical bone. Further, MAGP1⌬ mice have significantly less trabecular bone, the trabecular microarchitecture is more fragmented, and the diaphyseal cross-sectional area is significantly reduced. The remodeling defect seen in MAGP1⌬ mice is likely not due to an osteoblast defect, because MAGP1⌬ bone marrow stromal cells undergo osteoblastogenesis and form mineralized nodules. In vivo, MAGP1⌬ mice exhibit normal osteoblast number, mineralized bone surface, and bone formation rate. Instead, our findings suggest increased bone resorption is responsible for the osteopenia. The number of osteoclasts derived from MAGP1⌬ bone marrow macrophage cells is increased relative to the wild type, and osteoclast differentiation markers are expressed at earlier time points in MAGP1⌬ cells. In vivo, MAGP1⌬ mice have more osteoclasts lining the bone surface.RANKL(receptoractivatorofNF-Bligand)expressionissignificantly higher in MAGP1⌬ bone, and likely contributes to enhanced osteoclastogenesis. However, bone marrow macrophage cells from MAGP1⌬ mice show a higher propensity than do wildtype cells to differentiate to osteoclasts in response to RANKL, suggesting that they are also primed to respond to osteoclast-promoting signals. Together, our findings suggest that MAGP1 is a regulator of bone remodeling, and its absence results in osteopenia associated with an increase in osteoclast number.Originally thought to serve a purely structural role, the extracellular matrix (ECM) 2 is now known to be an important regulator of tissue development and homeostasis. Microfibrils are an abundant component of the ECM and can be found alone as microfibril bundles or associated with elastin in elastic fibers. Three fibrillins (FBN1, -2, and -3) provide the major structural components of these 10 nm diameter fibrils (1-3), although numerous microfibril-associated proteins interact with fibrillin and contribute to microfibril function. Fibrillin expression is widespread throughout development and is a product of most mesenchymal/interstitial cells (4). In the developing skeletal system, fibrillin expression has been documented in limb bud development, as well as in the adult bone (1, 5). Within the bone, fibrillin microfibrils can be found in the periosteal matrix, surrounding osteocytes, chondrocytes, and osteons, on the endochondral surface, and within the trabecular matrix (7). Microfibrils have clinical significance as mutations in the fibrillin genes give rise to a number of heritable connective tissue disorders. Mutations in the gene for fibrillin-1, for example, are linked to M...

Many preclinical evaluations of autologous small-diameter tissue-engineered vascular grafts (TEVGs) utilize cells from healthy humans or animals. However, these models hold minimal relevance for clinical translation, as the main targeted demographic is patients at high cardiovascular risk such as individuals with diabetes mellitus or the elderly. Stem cells such as adipose-derived mesenchymal stem cells (AD-MSCs) represent a clinically ideal cell type for TEVGs, as these can be easily and plentifully harvested and offer regenerative potential. To understand whether AD-MSCs sourced from diabetic and elderly donors are as effective as those from young nondiabetics (i.e., healthy) in the context of TEVG therapy, we implanted TEVGs constructed with human AD-MSCs from each donor type as an aortic interposition graft in a rat model. The key failure mechanism observed was thrombosis, and this was most prevalent in grafts using cells from diabetic patients. The remainder of the TEVGs was able to generate robust vascular-like tissue consisting of smooth muscle cells, endothelial cells, collagen, and elastin. We further investigated a potential mechanism for the thrombotic failure of AD-MSCs from diabetic donors; we found that these cells have a diminished potential to promote fibrinolysis compared to those from healthy donors. Together, this study served as proof of concept for the development of a TEVG based on human AD-MSCs, illustrated the importance of testing cells from realistic patient populations, and highlighted one possible mechanistic explanation as to the observed thrombotic failure of our diabetic AD-MSC-based TEVGs.

Vascular tissue engineering is an area of regenerative medicine that attempts to create functional replacement tissue for defective segments of the vascular network. One approach to vascular tissue engineering utilizes seeding of biodegradable tubular scaffolds with stem (and/or progenitor) cells wherein the seeded cells initiate scaffold remodeling and prevent thrombosis through paracrine signaling to endogenous cells. Stem cells have received an abundance of attention in recent literature regarding the mechanism of their paracrine therapeutic effect. However, very little of this mechanistic research has been performed under the aegis of vascular tissue engineering. Therefore, the scope of this review includes the current state of TEVGs generated using the incorporation of stem cells in biodegradable scaffolds and potential cell-free directions for TEVGs based on stem cell secreted products. The current generation of stem cell-seeded vascular scaffolds are based on the premise that cells should be obtained from an autologous source. However, the reduced regenerative capacity of stem cells from certain patient groups limits the therapeutic potential of an autologous approach. This limitation prompts the need to investigate allogeneic stem cells or stem cell secreted products as therapeutic bases for TEVGs. The role of stem cell derived products, particularly extracellular vesicles (EVs), in vascular tissue engineering is exciting due to their potential use as a cell-free therapeutic base. EVs offer many benefits as a therapeutic base for functionalizing vascular scaffolds such as cell specific targeting, physiological delivery of cargo to target cells, reduced immunogenicity, and stability under physiological conditions. However, a number of points must be addressed prior to the effective translation of TEVG technologies that incorporate stem cell derived EVs such as standardizing stem cell culture conditions, EV isolation, scaffold functionalization with EVs, and establishing the therapeutic benefit of this combination treatment.

Autologous tissue-engineered blood vessels (TEBVs) generated using adult stem cells have shown promising results, but many preclinical evaluations do not test the efficacy of stem cells from patient populations likely to need therapy (i.e., elderly and diabetic humans). Two critical functions of these cells will be (i) secreting factors that induce the migration of host cells into the graft and (ii) differentiating into functional vascular cells themselves. The purpose of this study was to analyze whether adipose-derived mesenchymal stem cells (AD-MSCs) sourced from diabetic and elderly patients have a reduced ability to promote human smooth muscle cell (SMC) migration and differentiation potential toward SMCs, two important processes in stem cell-based tissue engineering of vascular grafts. SMC monolayers were disrupted in vitro by a scratch wound and were induced to close the wound by exposure to media conditioned by AD-MSCs from healthy, elderly, and diabetic patients. Media conditioned by AD-MSCs from healthy patients promoted the migration of SMCs and did so in a dose-dependent manner; heating the media to 56°C eliminated the media's potency. AD-MSCs from diabetic and elderly patients had a decreased ability to differentiate into SMCs under angiotensin II stimulation; however, only AD-MSCs from elderly donors were unable to promote SMC migration. Gender and body-mass index of the patients showed no effect on either critical function of AD-MSCs. In conclusion, AD-MSCs from elderly patients may not be suitable for autologous TEBVs due to inadequate promotion of SMC migration and differentiation.