Understanding how relevant cell types respond to wear particles will reveal new avenues for treating osteolysis following joint replacements. In this study, we investigate the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on preosteoblast migration and function. We infused UHMWPE particles or saline into the left femur of mice and injected luciferase-expressing preosteoblasts (MC3T3 cells) into each left ventricle. Bioluminescence imaging (BLI) confirmed systemic administration of MC3T3 cells. BLI throughout the 28-day experiment showed greater MC3T3 migration to the site of particle infusion than to the site of saline infusion, with significant differences on days 0, 4, and 6 (p≤0.055). Immunostaining revealed a greater number of osteoblasts and osteoclasts in the particle-infused femora, indicating greater bone turnover. The bone mineralization of the particle-infused femora increased significantly when compared to saline-infused femora (an increase of 146.4±27.9 vs. 12.8±8.7 mg/mL, p=0.008). These results show that infused preosteoblasts can migrate to the site of wear particles. Additionally, as the migrated cells were associated with increased bone mineralization in spite of the presence of particles, increasing osteoblast recruitment is a potential strategy for combating bone loss due to increased osteoclast/macrophage number and decreased osteoblast function.
Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (∼92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.
Transforming growth factor-β1 (TGF-β1), an important cytokine with multiple functions, is secreted during wound healing. Previous studies have utilized two-dimensional (2D) cell culture to elucidate the functions of TGF-β1; however, 2D culture does not represent the complex three-dimensional (3D) in vivo environment. Using a synthetic hyaluronan (HA) extracellular matrix (ECM) hydrogel, we investigated the effect of TGF-β1 on fibroblasts cultured in three conditions—on tissue culture polystyrene (TCP), on HA (2D), and in HA (3D). After TGF-β1 treatment (0.1 to 20 ng/mL), morphological features and ECM regulation were analyzed by immunocytochemistry, Western blot, quantitative polymerase chain reaction, and zymogram assays. On TCP, cells showed the typical spindle shape with strong alpha smooth muscle actin (α-SMA) staining of cytoplasmic myofilaments along the cell axes after TGF-β1 treatment; on HA (2D), spindle-shape cells showed little α-SMA staining; in HA (3D), cells were smaller and rounded with less α-SMA deposition. The α-SMA gene and protein expression on TCP were significantly upregulated by TGF-β1, but TGF-β1 did not induce α-SMA expression in the presence of HA (both 2D and 3D). 3D HA culture significantly downregulated collagen I, III, and fibronectin expression, increased matrix metalloproteinase 1 and 2 (MMP1/MMP2) activity, upregulated MMP1 mRNA and downregulated TIMP3 mRNA expression. This study suggested that exogenous HA, particularly in 3D culture, appears to suppress ECM production, enhances ECM degradation and remodeling, and inhibits myofibroblast differentiation without decreasing TGF-β receptor expression.
To engineer tissue-like structures, cells must organize themselves into three-dimensional (3D) networks that mimic the native tissue microarchitecture. Microfabricated hydrogel substrates provide a potentially useful platform for directing cells into biomimetic tissue architecture in vitro. Here, we present microgrooved methacrylated gelatin hydrogels as a suitable platform to build muscle-like fibrous structures in a facile and highly reproducible fashion. Microgrooved hydrogel substrates with two different ridge sizes (50 and 100 μm) were fabricated to assess the effect of the distance between engineered myofibers on the orientation of the bridging C2C12 myoblasts and the formation of the resulting multinucleated myotubes. It was shown that although the ridge size did not significantly affect the C2C12 myoblast alignment, the wider-ridged micropatterned hydrogels generated more myotubes that were not aligned to the groove direction as compared to those on the smaller-ridge micropatterns. We also demonstrated that electrical stimulation improved the myoblast alignment and increased the diameter of the resulting myotubes. By using the microstructured methacrylated gelatin substrates, we built free-standing 3D muscle sheets, which contracted when electrically stimulated. Given their robust contractility and biomimetic microarchitecture, engineered tissues may find use in tissue engineering, biological studies, high-throughput drug screening, and biorobotics.
Hyaluronic acid (HA) is an extracellular matrix molecule with multiple physical and biological functions found in many tissues, including cartilage. HA has been incorporated in a number of biomaterial and scaffold systems. Howegver, HA in the material may be difficult to control if it is not chemically modified and chemical modification of HA may negatively impact biological function. In this study, we developed a poly(ethylene glycol) hydrogel with noncovalent HA-binding capabilities and evaluated its ability to support cartilage formation in vitro and in an articular defect model. Chondrogenic differentiation of mesenchymal stem cells encapsulated in the HA-interactive scaffolds containing various amounts of exogenous HA was evaluated. The HA-binding hydrogel without exogenous HA produced the best cartilage as determined by biochemical content (glysocaminoglycan and collagen), histology (Safranin O and type II collagen staining), and gene expression analysis for aggrecan, type I collagen, type II collagen, and sox-9. This HA-binding formulation was then translated to an osteochondral defect model in the rat knee. After 6 weeks, histological analysis demonstrated improved cartilage tissue production in defects treated with the HA-interactive hydrogel compared to noninteractive control scaffolds and untreated defects. In addition to the tissue repair in the defect space, the Safranin O staining in cartilage tissue surrounding the defect was greater in treatment groups where the HA-binding scaffold was applied. In sum, incorporation of a noncovalent HA-binding functionality into biomaterials provides an ability to interact with local or exogenous HA, which can then impact tissue remodeling and ultimately new tissue production.
Repair and regeneration of critical sized defects via the utilization of polymeric bone graft substitutes are challenges. Here, we introduce radially and axially graded multizonal bone graft substitutes fabricated from polycaprolactone (PCL), and PCL biocomposites with osteoconductive particles, that is, hydroxyapatite (HA), and β-tricalcium phosphate (TCP). The novel bone graft substitutes should provide a greater degree of freedom to the orthopedic surgeon especially for repair of critically sized bone defects. The modulus of the graft substitute could be tailored in the axial direction upon the systematic variation of the HA/TCP concentration, while in the radial direction the bone graft substitute consisted of an outer layer with high stiffness, encapsulating a softer core with greater porosity. The biocompatibility of the bone graft substitutes was investigated using in vitro culturing of human bone marrow-derived stromal cells followed by the analysis of cell proliferation and differentiation rates. The characterization of the tissue constructs included the enzymatic alkaline phosphates (ALP) activity, microcomputed tomography imaging, and polymerase chain reaction analysis involving the expressions of bone markers, that is, Runx2, ALP, collagen type I, osteopontin, and osteocalcin, overall demonstrating the differentiation of bone marrow derived stem cells (BMSCs) via osteogenic lineage and formation of mineralized bone tissue.
Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies.
A significant barrier to the success of engineered tissues is the inadequate transport of nutrients and gases to, and waste away from, cells within the constructs, after implantation. Generation of microtubular networks by endothelial cells in engineered constructs to mimic the in vivo transport scheme is essential for facilitating tissue survival by promoting the in vitro formation of microvessels that integrate with host microvasculature, after implantation. Previously, we reported that select pressures stimulate endothelial proliferation involving protubulogenic molecules such as fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-C (VEGF-C). Based on this, we investigated fluid pressure as a selective modulator of early tubulogenic activity with the intent of assessing the potential utility of this mechanical stimulus as a tissue-engineering control parameter. For this purpose, we used a custom pressure system to expose two-dimensional (2D) and three-dimensional (3D) cultures of endothelial cells to static pressures of 0 (controls), 20, or 40 mmHg for 3 days. Compared to controls, 2D endothelial cultures exposed to 20, but not 40 mmHg, exhibited significantly (p<0.05) enhanced cell growth that depended on VEGF receptor-3 (VEGFR-3), a receptor for VEGF-C. Moreover, endothelial cells grown on microbeads and suspended in 3D collagen gels under 20 mmHg, but not 40 mmHg, displayed significantly (p<0.05) increased sprout formation. Interestingly, pressure-dependent proliferation and sprout formation occurred in parallel with pressure-sensitive upregulation of VEGF-C and VEGFR-3 expression and were sensitive to local FGF-2 levels. Collectively, the results of the present study provided evidence that early endothelial-related tubulogenic activity depends on local hydrostatic pressure levels in the context of local growth factor conditions. In addition to relevance to microvascular diseases associated with interstitial hypertension (e.g., cancer and glaucoma), these findings provided first insight into the potential utility of hydrostatic pressure as a fine-tune control parameter to optimize microvascularization of tissue-engineering constructs in the in vitro setting before their implantation.
(1,3)-(1,6)-β-D-glucan (BG), a natural product of glucose polymers, has immune stimulatory activity that is especially effective in wound healing. In this study, poly(lactic-co-glycolic acid) (PLGA) membranes containing BGs (BG/PLGA membranes) were investigated for their wound-healing effects. The growth rate of human dermal fibroblasts was enhanced in BG/PLGA membranes. Their growth rates were improved with the increase of BG concentration in the membranes. The PLGA membranes with and without BGs were treated in full-thickness skin wound using male BALB/c nude mice (n=6 for each group). According to the animal study, BG/PLGA membranes enhanced the interaction with the surrounding cells in wound sites. In the wound site treated BG/PLGA, the positive of the Ki-67 (a proliferation cell marker) and the CD 31 (an endothelial cell marker) were 77.2%±5.6% and 34±8.6 capillaries. In the wound site treated PLGA, the Ki-67 positive cells were 51.3%±7.0%, and the positive-stained capillaries of CD 31 were 22.7±8.6. The wound site treated with BG/PLGA membranes was stronger stained of them in the wound site than those of the wound sites treated with PLGA membranes. BG/PLGA membranes accelerated wound healing by improving the interaction, proliferation of cells, and angiogenesis. BG/PLGA membranes can be useful as a skin substitute for enhancing wound healing.
Bone marrow-derived mesenchymal stem cells (BM-MSC) can be differentiated into lung epithelial-like cells (MSC-EC) in vitro. The response of BM-MSC and MSC-EC to stimuli may vary because of their character and differentiation. We aimed to investigate the factors that may influence in vitro differentiation of BM-MSC to MSC-EC. We determined the response of BM-MSC, MSC-EC, bronchial epithelial cells, and alveolar epithelial cells to tumor necrosis factor (TNF)-α stimulation. We also investigated the changes in micro(mi)RNA-146a, miRNA-155, and TNF receptor 1 (TNFR1) expression after stimulation. Our results demonstrate that the addition of transforming growth factor-β1 and extracellular matrix collagen are required to facilitate such differentiation. After 3 weeks of culture, the morphological appearance and expression of airway epithelial markers, cytokeratin and Clara cell secretory protein, in MSC-EC were characteristics of lung epithelial cells. In response to TNF-α stimulation, the maximal interleukin (IL)-8 production by BM-MSC at the 24-h time point was 4.8 times greater compared with MSC-EC. TNF-α induced a significant increase in the expression of miRNA-146a in BM-MSC as compared with MSC-EC. miRNA-155 expression remained unchanged after stimulation. TNFR1 mRNA also significantly increased in BM-MSC after TNF-α stimulation. This was not observed in MSC-EC. Transfection with miRNA-146a mimics resulted in a significant increase of miRNA-146a expression and IL-8 production in both types of cells. In contrast, miRNA-146a inhibitors reduced miRNA-146a expression and IL-8 production. Overexpression of miRNA-146a, which positively regulates TNF-α-induced IL-8 release, may enhance the inflammatory response in both BM-MSC and MSC-EC. The expression of miRNA-146a and the response to stimuli may be modulated through mature differentiation of BM-MSC.
Fibroblast growth factor-2 (FGF-2) is a well-characterized protein that is used in the treatment of healing-impaired wounds. We previously reported that fragmin/protamine microparticles (F/P MPs) are useful as biodegradable carriers for the controlled release of cytokines. We examined the ability of FGF-2-containing (FGF-2/) F/P MPs to prevent limb loss in an experimentally induced ischemic hindlimb model using adult Balb/c-nu/nu male mice. One day after inducing ischemia, intramuscular injections of 100 μL of FGF-2/F/P MPs turbid suspension (10 μg/mL FGF-2 and 6 mg/mL F/P MPs) were administered into eight sites of the ischemic hindlimb. A 100-μL suspension of each of the following—10 μg/mL FGF-2, 6 mg/mL F/P MPs, and phosphate-buffered saline (PBS; the control)—was similarly injected into the hindlimb. From 5 days onward after the injections, recovery from ischemia was observed in the FGF-2/F/P MP-treated group, but only partial recovery occurred in the FGF-2-treated group. The F/P MP-treated and PBS-treated groups (i.e., control) exhibited no recovery from the ischemia. The histological evaluations of the hindlimbs also confirmed that the capillary (i.e., mature vessels) density was significantly higher in the FGF-2/F/P MP-treated group than in the other groups. The mice injected with FGF-2/F/P MPs also recovered hindlimb blood flow, as reflected by oxygen saturation and surface temperature evaluation. Our present approach using FGF-2/F/P MPs could be considered a valuable option for the therapeutic treatment of peripheral ischemic diseases.
Low back pain is a leading health problem in the United States, which is most often resulted from nucleus pulposus (NP) degeneration. To date, the replacement of degenerated NP relies entirely on mechanical devices. However, a biological NP replacement implant is more desirable. Here, we report the regeneration of NP tissue using a biodegradable nanofibrous (NF) scaffold. Rabbit NP cells were seeded on the NF scaffolds to regenerate NP-like tissue both in vitro and in a subcutaneous implantation model. The NP cells on the NF scaffolds proliferated faster than those on control solid-walled (SW) scaffolds in vitro. Significantly more extracellular matrix (ECM) production (glycosaminoglycan and type II collagen) was found on the NF scaffolds than on the control SW scaffolds. The constructs were then implanted in the caudal spine of athymic rats for up to 12 weeks. The tissue-engineered NP could survive, produce functional ECM, remain in place, and maintain the disc height, which is similar to the native NP tissue.
Our laboratory has developed a tensile culture bioreactor as a system for understanding mesenchymal stem cell (MSC) differentiation toward a tendon/ligament fibroblast phenotype in response to cyclic tensile strain. In this study, we investigated whether increased degradability of the biomaterial carrier would induce changes in MSC morphology and subsequent upregulation of tendon/fibroblast markers under tensile strain. Degradability of a synthetic poly(ethylene glycol) hydrogel was introduced by incorporating either fast- or slow-degrading matrix metalloproteinase (MMP)–sensitive peptide sequences into the polymer backbone. Although a decline in cellularity was observed over culture in all sample groups, at 14 days, MSCs were significantly more spread in fast-cleaving gels (84%±8%) compared with slow-cleaving gels (59%±4%). Cyclic tensile strain upregulated tendon/ligament fibroblast-related genes, such as collagen III (3.8-fold vs. 2.1-fold in fast-degrading gels) and tenascin-C (2.5-fold vs. 1.7-fold in fast-degrading gels). However, few differences were observed in gene expression between different gel types. Immunostaining demonstrated increased collagen III deposition in dynamically strained gels at day 14, as well as increased collagen I and tenascin-C deposition at day 14 in all groups. Results suggest that cell spreading may not be a major factor controlling MSC response to cyclic strain in this system over 14 days. However, these findings provide key parameters for the design of future biomaterial carriers and strain regimens to prime stem cells to a tendon/ligament phenotype prior to release and use in vivo.
Cryopreservation is important for clinical translation of tissue-engineered constructs. With respect to a pancreatic substitute, encapsulated islets or beta cells have been widely studied for the treatment of insulin-dependent diabetes mellitus. Besides cell viability loss, cryopreservation may affect the function of the remaining viable cells in a pancreatic substitute by altering fundamental processes in glucose-stimulated insulin secretion, such as pathways associated with intermediary metabolism, potentially leading to insulin-secretion defects. In this study, we used 13C nuclear magnetic resonance (NMR) spectroscopy and isotopomer analysis to determine the effects of conventional freezing and ice-free cryopreservation (vitrification) on carbon flow through tricarboxylic acid (TCA) cycle–associated pathways in encapsulated murine insulinoma βTC-tet cells; the secretory function of the encapsulated cells postpreservation was also evaluated. Specifically, calcium alginate–encapsulated βTC-tet cells were frozen or vitrified with a cryoprotectant cocktail. Beads were warmed and 13C labeling and extraction were performed. Insulin secretion rates were determined during basal and labeling periods and during small-scale glucose stimulation and K+-induced depolarization. Relative metabolic fluxes were determined from 13C NMR spectra using a modified single pyruvate pool model with the tcaCALC modeling program. Treatments were compared with nonpreserved controls. Results showed that relative carbon flow through TCA-cycle-associated pathways was not affected by conventional freezing or vitrification. However, vitrification, but not freezing, led to impaired insulin secretion on a per viable cell basis. The reduced secretion from the Vitrified group occurred irrespective of scale and was present whether secretion was stimulated by glucose or K+-induced depolarization, indicating that it might be due to a defect in late-stage secretion events.
Since robust osteogenic differentiation and mineralization are integral to the engineering of bone constructs, understanding the impact of the cellular microenvironments on human mesenchymal stem cell (hMSCs) osteogenic differentiation is crucial to optimize bioreactor strategy. Two perfusion flow conditions were utilized in order to understand the impact of the flow configuration on hMSC construct development during both pre-culture (PC) in growth media and its subsequent osteogenic induction (OI). The media in the in-house perfusion bioreactor was controlled to perfuse either around (termed parallel flow [PF]) the construct surfaces or penetrate through the construct (termed transverse flow [TF]) for 7 days of the PC followed by 7 days of the OI. The flow configuration during the PC not only changed growth kinetics but also influenced cell distribution and potency of osteogenic differentiation and mineralization during the subsequent OI. While shear stress resulted from the TF stimulated cell proliferation during PC, the convective removal of de novo extracellular matrix (ECM) proteins and growth factors (GFs) reduced cell proliferation on OI. In contrast, the effective retention of de novo ECM proteins and GFs in the PC constructs under the PF maintained cell proliferation under the OI but resulted in localized cell aggregations, which influenced their osteogenic differentiation. The results revealed the contrasting roles of the convective flow as a mechanical stimulus, the redistribution of the cells and macromolecules in 3D constructs, and their divergent impacts on cellular events, leading to bone construct formation. The results suggest that the modulation of the flow configuration in the perfusion bioreactor is an effective strategy that regulates the construct properties and maximizes the functional outcome.
Ischemic preconditioning (IPC) is a potent and effective means of protecting cells against ischemic injury. The protection has been demonstrated to involve release of paracrine factors that promote cell survival and angiogenesis, factors important for successful tissue engineering. The aim of the present study was to determine whether IPC of a vascular bed in vivo is an effective strategy to prepare it for tissue engineering with implanted cells. To test this hypothesis, an in vivo vascularized tissue engineering approach was employed, whereby polyacrylic chambers were placed around the femoral vessels of adult Sprague-Dawley rats. IPC was induced by 3 cycles of 5 min femoral artery occlusion interspersed with 5-min periods of reperfusion. Rats subjected to IPC generated bigger tissue constructs at 7 and 28 days postimplantation of empty chambers (∼50% increase in weight and volume, p<0.05). Morphometric counting of Masson trichrome stained tissue sections revealed significantly greater tissue construct volumes in ischemic preconditioned vascular beds at 7 and 28 days, increasing both fibrin matrix and vascularized tissue. Furthermore, morphometry of lectin-labeled blood vessels indicated an increase in vascular volume in IPC tissue constructs (∼100% increase vs. control, p<0.05). To investigate the cytoprotective effect of IPC, we implanted DiI-labeled neonatal rat cardiomyocytes in the chambers for 3 days, and IPC significantly reduced apoptosis of implanted cells as determined by the TUNEL assay and cleaved caspase-3 immunostaining. Furthermore, IPC significantly increased the cardiac muscle volume and vascular volume at 28 days after implantation of cardiomyocytes. In conclusion, in vivo IPC promotes survival of implanted cardiomyocytes and is associated with enhanced angiogenesis. IPC may represent a new approach to optimize tissue engineering with implanted cells.
Topographic features at the dermal–epidermal junction (DEJ) provide instructive cues critical for modulating keratinocyte functions and enhancing the overall architecture and organization of skin. This interdigitated interface conforms to a series of rete ridges and papillary projections on the dermis that provides three-dimensional (3D) cellular microenvironments as well as structural stability between the dermal and epidermal layers during mechanical loading. The dimensions of these cellular microenvironments exhibit regional differences on the surface of the body, and quantitative histological analyses have shown that localization of highly proliferative keratinocytes also varies, according to the regional geometries of these microenvironments. In this study, we combined photolithography, collagen processing, and biochemical conjugation techniques to create microfabricated dermal epidermal regeneration matrices (μDERMs) with features that mimic the native 3D cellular microenvironment at the DEJ. We used this model system to study the effect of the 3D cellular microenvironment on epithelialization and basal keratinocyte interaction with the microenvironment on the surface of the μDERMs. We found that features closely mimicking those in high-friction areas of the body (deep, narrow channels) epithelialized faster than features mimicking low-friction areas. Additionally, when evaluating β1 expression, an integrin involved in epidermal morphogenesis, it was found that integrin-bright expression was localized in the depths of the features, suggesting that the μDERMs may play a role in defining cellular microenvironments as well as a protective environment for the regenerative population of keratinocytes. The outcomes of this study suggest that μDERMs can serve as a robust biomimetic model system to evaluate the roles of the 3D microenvironment on enhancing the regenerative capacity and structural stability of bioengineered skin substitutes.
As a common feature of many neurological diseases and injury, the loss of axon pathways can have devastating effects on function. Here, we demonstrate a new strategy to restore damaged axon pathways using transplantable miniature constructs consisting of living neurons and axonal tracts internalized within hydrogel tubes. These hydrogel microconduits were developed through an iterative process to support neuronal survival and directed axon growth. The design included hollow agarose tubes providing a relatively stiff outer casing to direct constrained unidirectional outgrowth of axons through a central soft collagen matrix, with overall dimensions of 250 μm inner diameter ×500 μm outer diameter and extending up to several centimeters. The outer casing was also designed to provide structural support of neuronal/axonal cultures during transplantation of the construct. Using neuron culture conditions optimized for the microconduits, dissociated dorsal root ganglia neurons were seeded in the collagen at one end of the conduits. Over the following week, high-resolution confocal microscopy demonstrated that the neurons survived and the somata remained in a tight cluster at the original seeding site. In addition, robust outgrowth of axons from the neurons was found, with axon fascicles constrained in a longitudinal projection along the internal collagen canal and extending over 5 mm in length. Notably, this general geometry recapitulates the anatomy of axon tracts. As such, these constructs may be useful to repair damaged axon projections by providing a transplantable bridge of living axons. Moreover, the small size of the construct permits follow-on studies of minimally invasive transplantation into potentially sensitive regions of the nervous system.
Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be obtained without an invasive surgery. To date, there has been no report on seeding hUCMSCs in three-dimensional scaffolds for muscle tissue engineering. The objectives of this study were to (1) investigate hUCMSC seeding in a scaffold for muscle engineering and (2) develop a novel construct consisting of hUCMSC-encapsulating and fast-degradable microbeads inside a hydrogel matrix. The rationale was that the hydrogel matrix would maintain the defect volume, while the microbeads would degrade to release the cells and concomitantly create macropores in the matrix. hUCMSCs were encapsulated in alginate-fibrin microbeads, which were packed in an Arg-Gly-Asp (RGD)-modified alginate matrix (AM). This construct is referred to as hUCMSC-microbead-AM. The control consisted of the usual cell encapsulation in AM without microbeads (referred to as hUCMSC-AM). In the hUCMSC-AM construct, the hUCMSCs showed as round dots with no spreading at 1–14 days. In contrast, cells in the hUCMSC-microbead-AM construct had a healthy spreading and elongated morphology. The microbeads successfully degraded and released the cells at 8 days. Myogenic expressions for hUCMSC-microbead-AM were more than threefold those of hUCMSC-AM (p<0.05). Immunofluorescence for myogenic markers was much stronger for hUCMSC-microbead-AM than hUCMSC-AM. Muscle creatine kinase of hUCMSC-microbead-AM at 14 days was twofold that of hUCMSC-AM (p<0.05). In conclusion, hUCMSC encapsulation in novel fast-degradable microbeads inside a hydrogel matrix was investigated for muscle engineering. Compared to the usual method of seeding cells in a hydrogel matrix, hUCMSC-microbead-AM construct had greatly improved cell viability and myogenic differentiation, and hence, is promising to enhance muscle regeneration.
Autologous chondrocyte implantation is an effective treatment for damaged articular cartilage. However, this method involves surgical procedures that may cause further cartilage degeneration, and in vitro expansion of chondrocytes can result in dedifferentiation. Adipose-derived stem cells (ADSCs) may be an alternative autologous cell source for cartilage regeneration. In this study, we developed an effective method for large-scale in vitro chondrogenic differentiation, which is the procedure that would be required for clinical applications, and the subsequent in vivo cartilage formation of human ADSCs (hADSCs). The spheroid formation and chondrogenic differentiation of hADSCs were induced on a large scale by culturing hADSCs in three-dimensional suspension bioreactors (spinner flasks). In vitro chondrogenic differentiation of hADSCs was enhanced by a spheroid culture compared with a monolayer culture. The enhanced chondrogenesis was probably attributable to hypoxia-related cascades and enhanced cell–cell interactions in hADSC spheroids. On hADSCs loading in fibrin gel and transplantation into subcutaneous space of athymic mice for 4 weeks, the in vivo cartilage formation was enhanced by the transplantation of spheroid-cultured hADSCs compared with that of monolayer-cultured hADSCs. This study shows that the spheroid culture may be an effective method for large-scale in vitro chondrogenic differentiation of hADSCs and subsequent in vivo cartilage formation.
Volumetric muscle loss (VML) resulting from traumatic accidents, tumor ablation, or degenerative disease is associated with limited treatment options and high morbidity. The lack of a reliable and reproducible animal model of VML has hindered the development of effective therapeutic strategies. The present study describes a critical-sized excisional defect within the mouse quadriceps muscle that results in an irrecoverable volumetric defect. This model of VML was used to evaluate the efficacy of a surgically placed inductive biologic scaffold material composed of porcine small intestinal submucosa–extracellular matrix (SIS-ECM). The targeted placement of an SIS-ECM scaffold within the defect was associated with constructive tissue remodeling including the formation of site-appropriate skeletal muscle tissue. The present study provides a reproducible animal model with which to study VML and shows the therapeutic potential of a bioscaffold-based regenerative medicine approach to VML.
The successful use of transplanted cells and/or growth factors for tissue repair is limited by a significant cell loss and/or rapid growth factor diffusion soon after implantation. Highly porous alginate scaffolds formed with covalent crosslinking have been used to improve cell survival and growth factor release kinetics, but require open-wound surgical procedures for insertion and have not previously been designed to readily degrade in vivo. In this study, a biodegradable, partially crosslinked alginate scaffold with shape-memory properties was fabricated for minimally invasive surgical applications. A mixture of high and low molecular weight partially oxidized alginate modified with RGD peptides was covalently crosslinked using carbodiimide chemistry. The scaffold was compressible 11-fold and returned to its original shape when rehydrated. Scaffold degradation properties in vitro indicated ∼85% mass loss by 28 days. The greater than 90% porous scaffolds released the recombinant growth factor insulin-like growth factor-1 over several days in vitro and allowed skeletal muscle cell survival, proliferation, and migration from the scaffold over a 28-day period. The compressible scaffold thus has the potential to be delivered by a minimally invasive technique, and when rehydrated in vivo with cells and/or growth factors, could serve as a temporary delivery vehicle for tissue repair.
The ideally engineered bone should have similar structural and functional properties to the native tissue. Although structural integrity is critical for functional bone regeneration, we know less about modulating the structural properties of the engineered bone elicited by bone morphogenetic protein (BMP) than efficacy and safety. Erythropoietin (Epo), a primary erythropoietic hormone, has been used to augment blood transfusion in orthopedic surgery. However, the effects of Epo on bone regeneration are not well known. Here, we determined the role of Epo in BMP2-induced bone regeneration using a cranial defect model. Epo administration improved the quality of BMP2-induced bone and more closely resembled natural cranial bone with a higher bone volume (BV) fraction and lower marrow fraction when compared with BMP2 treatment alone. Epo increased red blood cells (RBCs) in peripheral blood and also increased hematopoietic and mesenchymal stem cell (MSC) populations in bone marrow. Consistent with our previous work, Epo increased osteoclastogenesis both in vitro and in vivo. Results from a metatarsal organ culture assay suggested that Epo-promoted osteoclastogenesis contributed to angiogenesis because angiogenesis was blunted when osteoclastogenesis was blocked by alendronate (ALN) or osteoprotegerin (OPG). Earlier calcification of BMP2-induced temporary chondroid tissue was observed in the Epo+BMP group compared to BMP2 alone. We conclude that Epo significantly enhanced the outcomes of BMP2-induced cranial bone regeneration in part through its actions on osteoclastogenesis and angiogenesis.
In bone tissue engineering, growth factors are widely used. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are the most well-known regulators of osteogenesis and angiogenesis. We investigated whether the timing of dual release of VEGF and BMP-2 influences the amount of bone formation in a large-animal model. Poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were loaded with BMP-2 or VEGF to create sustained-release profiles, and rapidly degrading gelatin was loaded with either growth factor for fast-release profiles. To study in vivo osteogenicity, the two delivery vehicles were combined with biphasic calcium phosphate (BCP) scaffolds and implanted in 10 Beagle dogs for 9 weeks, at both ectopic (paraspinal muscles) and orthotopic sites (critical-size ulnar defect). The 9 ectopic groups contained combined or single BMP/VEGF dosage, in sustained- or fast-release profiles. In the ulnae of 8 dogs, fast VEGF and sustained BMP-2 were applied to one leg, and the other received the opposite release profiles. The two remaining dogs received bilateral control scaffolds. Bone growth dynamics was analyzed by fluorochrome injection at weeks 3, 5, and 7. Postoperative and posteuthanization X-rays of the ulnar implants were taken. After 9 weeks of implantation, bone quantity and bone growth dynamics were studied by histology, histomorphometry, and fluorescence microscopy. The release of the growth factors resulted in both enhanced orthotopic and ectopic bone formation. Bone formation started before 3 weeks and continued beyond 7 weeks. The ectopic BMP-2 fast groups showed significantly more bone compared to sustained release, independent of the VEGF profile. The ulna implants revealed no significant differences in the amount of bone formed. This study shows that timing of BMP-2 release largely determines speed and amount of ectopic bone formation independent of VEGF release. Furthermore, at the orthotopic site, no significant effect on bone formation was found from a timed release of growth factors, implicating that timed-release effects are location dependent.
Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. Previously, our studies have shown a significant mass of vascularized cardiac tissue can be generated using a vascularized tissue engineering chamber approach in nude rats. In this present study, syngeneic rats were investigated as an animal model for cardiac tissue engineering using the arteriovenous loop (AVL) chamber in the presence of a functional immune system. Neonatal cardiomyocytes implanted into the AVL chamber survived and assembled into a contractile flap confirming the basic features we previously showed in growing a cardiac construct. There was no significant loss of the assembled cardiac muscle from immune response. The engineered cardiac muscle flaps (ECMFs) formed were transplanted to the neck vessels of the same animal using a microsurgical technique, and all transplanted tissues remained contractile. The cardiac muscle volume of the control and transplant groups was estimated with histomorphometry using desmin and α-sarcomeric actin immunostaining, and there were no significant differences between the two groups. Finally, utilizing a novel model of transplantation, the ECMFs were transplanted to the heart of a recipient syngeneic rat as a vascularized tissue. The cardiac muscle within the transplanted ECMF was shown to survive and remain contractile for the 4-week post-transplantation period, and importantly, the cardiomyocytes retained the elongated, striated appearance of a mature phenotype. This study demonstrated the proof of concept for transplanting tissue-engineered cardiac muscle as a vascularized cardiac construct.