In vivo generation of a mature and functional artificial skeletal muscle.

Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro-generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG-fibrinogen hydrogel. Once engineered to express placental-derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG-fibrinogen-embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient-specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix.

Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 µg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects.

Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration.

BACKGROUND: Cell-transplantation therapies have attracted attention as treatments for skeletal-muscle disorders; however, such research has been severely limited by poor cell survival. Tissue engineering offers a potential solution to this problem by providing biomaterial adjuvants that improve survival and engraftment of donor cells. METHODS: In this study, we investigated the use of intra-muscular transplantation of mesoangioblasts (vessel-associated progenitor cells), delivered with an injectable hydrogel biomaterial directly into the tibialis anterior (TA) muscle of acutely injured or dystrophic mice. The hydrogel cell carrier, made from a polyethylene glycol-fibrinogen (PF) matrix, is polymerized in situ together with mesoangioblasts to form a resorbable cellularized implant. RESULTS: Mice treated with PF and mesoangioblasts showed enhanced cell engraftment as a result of increased survival and differentiation compared with the same cell population injected in aqueous saline solution. CONCLUSION: Both PF and mesoangioblasts are currently undergoing separate clinical trials: their combined use may increase chances of efficacy for localized disorders of skeletal muscle.

Regenerative medicine as applied to general surgery.

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

A combined cell therapy and in-situ tissue-engineering approach for myocardial repair.

Myocardial cell-replacement strategies are hampered by limited sources for human cardiomyocytes and by significant cell loss following transplantation. We tested the hypothesis that a combined delivery of cardiomyocytes with an in-situ polymerizable hydrogel into a post-MI rat heart will result in better functional outcomes than each intervention alone. A photopolymerizable, biodegradable, PEGylated-fibrinogen (PF) hydrogel matrix was used as the carrier for the cardiomyocytes [neonatal rat ventricular cardiomyocytes (NRVCMs) or human embryonic stem cell-derived cardiomyocytes (hESC-CMs)]. Infarcted rat hearts (LAD ligation) were randomized to injection of saline, NRVCMs, biopolymer, or combined biopolymer-cell delivery. Echocardiography revealed typical post-infarction remodeling after 30 days in the saline-injected control group [deterioration of fractional shortening (FS) by 31.0 ± 3.6%]. Injection of NRVCMs or PF alone significantly (p < 0.01) altered this remodeling process (slightly increasing FS by 3.1 ± 6.6% and 0.5 ± 5.3% respectively). Co-injection of the NRVCMs with PF matrix resulted in a significant increase in the cell-graft area (by 144%) and in the highest improvements in FS (by 26.3 ± 6.6%). Finally, feasibility studies were performed with the PF matrix and hESC-CMs. We conclude that an injectable in-situ forming hydrogel can act as a cardiomyocyte cell-carrier and add to the beneficial effects of the grafted cells in preventing unfavorable post-infarction cardiac remodeling.

Regenerative medicine as applied to solid organ transplantation: current status and future challenges.

In the last two decades, regenerative medicine has shown the potential for "bench-to-bedside" translational research in specific clinical settings. Progress made in cell and stem cell biology, material sciences and tissue engineering enabled researchers to develop cutting-edge technology which has lead to the creation of nonmodular tissue constructs such as skin, bladders, vessels and upper airways. In all cases, autologous cells were seeded on either artificial or natural supporting scaffolds. However, such constructs were implanted without the reconstruction of the vascular supply, and the nutrients and oxygen were supplied by diffusion from adjacent tissues. Engineering of modular organs (namely, organs organized in functioning units referred to as modules and requiring the reconstruction of the vascular supply) is more complex and challenging. Models of functioning hearts and livers have been engineered using "natural tissue" scaffolds and efforts are underway to produce kidneys, pancreata and small intestine. Creation of custom-made bioengineered organs, where the cellular component is exquisitely autologous and have an internal vascular network, will theoretically overcome the two major hurdles in transplantation, namely the shortage of organs and the toxicity deriving from lifelong immunosuppression. This review describes recent advances in the engineering of several key tissues and organs.

A photopolymerizable hydrogel for 3-D culture of human embryonic stem cell-derived cardiomyocytes and rat neonatal cardiac cells.

The purpose of this study was to assess the in vitro ability of two types of cardiomyocytes (cardiomyocytes derived from human embryonic stem cells (hESC-CM) and rat neonatal cardiomyocytes (rN-CM)) to survive and generate a functional cardiac syncytium in a three-dimensional in situ polymerizable hydrogel environment. Each cell type was cultured in a PEGylated fibrinogen (PF) hydrogel for up to two weeks while maturation and cardiac function were documented in terms of spontaneous contractile behavior and biomolecular organization. Quantitative contractile parameters including contraction amplitude and synchronization were measured by non-invasive image analysis. The rN-CM demonstrated the fastest maturation and the most significant spontaneous contraction. The hESC-CM maturation occurred between 10-14 days in culture, and exhibited less contraction amplitude and synchronization in comparison to the rN-CMs. The maturation of both cell types within the hydrogels was confirmed by cardiac-specific biomolecular markers, including alpha-sarcomeric actin, actinin, and connexin-43. Cellular responsiveness to isoproterenol, carbamylcholine and heptanol provided further evidence of the cardiac maturation in the 3-D PF hydrogel as well as identified a potential to use this system for in vitro drug screening. These findings indicate that the PF hydrogel biomaterial can be used as an in situ polymerizable biomaterial for stem cells and their cardiomyocyte derivatives.

Matrix stiffness affects spontaneous contraction of cardiomyocytes cultured within a PEGylated fibrinogen biomaterial.

Successful implementation of cardiac cell transplantation for treating damaged myocardium relies on the development of improved injectable biomaterials. A novel biomaterial technology using PEGylated fibrinogen has been developed with controllable physicochemical properties based on the poly(ethylene glycol) (PEG) constituent. In addition, the fibrinogen backbone of the material confers inherent bioactivity to cells. The purpose of this investigation was to explore by in vitro techniques the use of this biomaterial as a scaffold for cardiac tissue regeneration. To this end neonatal rat cardiomyocytes were cultivated in PEGylated fibrinogen constructs. The cell-seeding density and biomaterial composition were optimized to obtain maximum spontaneous contraction of the constructs. Quantitative characterization of the contraction pattern was accomplished by video image analysis. It was possible to demonstrate an inverse correlation between the material stiffness and the amplitude of contraction of the tissue constructs by changing the modulus of the matrix using different compositions of PEG and fibrinogen. The relationship between matrix stiffness, cell density and tissue contraction also provided some insight into the mechanism of cellular remodeling that ultimately leads to synchronized contraction of the constructs. These findings indicate that PEGylated fibrinogen hydrogels can be used as a scaffold for cardiomyocytes, and offer the possibility of controlling cellular remodeling via simple compositional modifications to the matrix.