New Anatomy: Plastinated Prosections And Slices Use Student Time Efficiently And Increase Enjoyment
[New York NY : NYU College of Dentistry. NYU Academy of Distinguished Educators], 2011
An improved collagen scaffold for skeletal regeneration
Bone repair and regeneration is one of the most extensively studied areas in the field of tissue engineering. All of the current tissue engineering approaches to create bone focus on intramembranous ossification, ignoring the other mechanism of bone formation, endochondral ossification. We propose to create a transient cartilage template in vitro, which could serve as an intermediate for bone formation by the endochondral mechanism once implanted in vivo. The goals of the study are (1) to prepare and characterize type I collagen sponges as a scaffold for the cartilage template, and (2) to establish a method of culturing chondrocytes in type I collagen sponges and induce cell maturation. Collagen sponges were generated from a 1% solution of type I collagen using a freeze/dry technique followed by UV light crosslinking. Chondrocytes isolated from two locations in chick embryo sterna were cultured in these sponges and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Material strength testing as well as microscopic and biochemical analyzes were conducted to evaluate the properties of sponges and cell behavior during the culture period. We found that our collagen sponges presented improved stiffness and supported chondrocyte attachment and proliferation. Cells underwent maturation, depositing an abundant extracellular matrix throughout the scaffold, expressing high levels of type X collagen, type I collagen and alkaline phosphatase. These results demonstrate that we have created a transient cartilage template with potential to direct endochondral bone formation after implantation.
Human satellite progenitor cells for use in myofascial repair: isolation and characterization
Current use of prosthetic meshes and implants for myofascial reconstruction has been associated with infectious complications, long-term failure, and dissatisfying cosmetic results. Our laboratory has developed a small animal model for ventral hernia repair, which uses progenitor cells isolated from a skeletal muscle biopsy. In the model, progenitor cells are expanded in vitro, seeded onto a nonimmunogenic, novel aligned scaffold of bovine collagen and placed into the defect as a living adjuvant to the innate repair mechanism. The purpose of the current investigation is to examine the feasibility of translating our current model to humans. As a necessary first step we present our study on the efficacy of isolating satellite cells from 9 human donor biopsies. We were able to successfully translate our progenitor cell isolation and culture protocols to a human model with some modifications. Specifically, we have isolated human satellite muscle cells, expanded them in culture, and manipulated these cells to differentiate into myotubes in vitro. Immunohistochemical analysis allowed the characterization of distinct progenitor cell cycle stages and quantification of approximate cell number. Furthermore, isolated cells were tracked via cytoplasmic nanocrystal labeling and observed using confocal microscopy.
Erratum: Focused in vivo genetic analysis of implanted engineered myofascial constructs (Journal of Investigative Surgery 22:1) [Correction]
Focused in vivo genetic analysis of implanted engineered myofascial constructs
Successfully engineering functional muscle tissue either in vitro or in vivo to treat muscle defects rather than using the host muscle transfer would be revolutionary. Tissue engineering is on the cutting edge of biomedical research, bridging a gap between the clinic and the bench top. A new focus on skeletal muscle tissue engineering has led investigators to explore the application of satellite cells (autologous muscle precursor cells) as a vehicle for engineering tissues either in vitro or in vivo. However, few skeletal muscle tissue-engineering studies have reported on successful generation of living tissue substitutes for functional skeletal muscle replacement. Our model system combines a novel aligned collagen tube and autologous skeletal muscle satellite cells to create an engineered tissue repair for a surgically created ventral hernia as previously reported [SA Fann, L Terracio, W Yan, et al., A model of tissue-engineered ventral hernia repair, J Invest Surg. 2006;19(3):193-205]. Several key features we specifically observe are the significant persistence of transplanted skeletal muscle cell mass within the engineered repair, the integration of new tissue with adjacent native muscle, and the presence of significant neovascularization. In this study, we report on our experience investigating the genetic signals important to the integration of neoskeletal muscle tissue. The knowledge gained from our model system applies to the repair of severely injured extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.
Tissue engineering of skeletal muscle
Loss of skeletal muscle profoundly affects the health and well-being of patients, and there currently is no way to replace lost muscle. We believe that a key step in the development of a prosthesis for reconstruction of dysfunctional muscular tissue is the ability to reconstitute the in vivo-like 3-dimensional (3D) organization of skeletal muscle in vitro with isolated satellite cells. In our present proof of principle studies, we have successfully constructed a multilayered culture of skeletal muscle cells, derived from neonatal satellite cells, that are distributed in a 3D pattern of organization that mimics many of the features of intact tissue. These multilayered cultures are composed of elongated multinucleated myotubes that are MyoD positive. Histological studies indicate that the multiple layers of myotubes can be distinguished. Expression of muscle-specific markers such as myosin heavy chain, dystrophin, integrin alpha-7, alpha-enolase, and beta-enolase was detected using real-time reverse transcriptase polymerase chain reaction at levels near adult values. Physiological measurements of the engineered skeletal muscle showed that they tetanize and display physiologic force length behavior, although developed force per cross-sectional area was below that of native rat skeletal muscle.
A model of tissue-engineered ventral hernia repair
We have developed a tissue-engineered ventral hernia repair system using our novel aligned collagen tube and autologous skeletal muscle satellite cells. In this model system, skeletal muscle satellite cells were isolated from a biopsy, expanded in culture, and incorporated into our collagen tube scaffold, forming the tissue-engineered construct. We characterized the results of the repaired hernias on both the gross and microscopic scales and compared them to an unrepaired control, an autologous muscle repair control, and a collagen-tube-only repair. Untreated animals developed a classic hernia sac, devoid of abdominal muscle and covered only with a thin layer of mesothelial tissue. Significant muscle, small-diameter blood vessels, and connective tissue were apparent in both the autologous control and the engineered muscle repairs. The engineered muscle repairs became cellularized, vascularized, and integrated with the native tissue, hence becoming a "living" repair. A tissue-engineered construct repair of ventral hernias with subsequent incorporation and vascularization could provide the ultimate in anterior wall myofascial defect repair and would further the understanding of striated muscle engineering. The knowledge gained from our model system would have immediate application to mangled extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.
The new anatomy: Dissectionless but not cadaverless [Meeting Abstract]
Skeletal muscle: in vitro growth with applications to tissue engineering [Meeting Abstract]
Cardiovascular tissue engineering I. Perfusion bioreactors: a review
Tissue engineering is a fast-evolving field of biomedical science and technology with future promise to manufacture living tissues and organs for replacement, repair, and regeneration of diseased organs. Owing to the specific role of hemodynamics in the development, maintenance, and functioning of the cardiovascular system, bioreactors are a fundamental of cardiovascular tissue engineering. The development of perfusion bioreactor technology for cardiovascular tissue engineering is a direct sequence of previous historic successes in extracorporeal circulation techniques. Bioreactors provide a fluidic environment for tissue engineered tissue and organs, and guarantee their viability, maturation, biomonitoring, testing, storage, and transportation. There are different types of bioreactors and they vary greatly in their size, complexity, and functional capabilities. Although progress in design and functional properties of perfusion bioreactors for tissue engineered blood vessels, heart valves, and myocardial patches is obvious, there are some challenges and insufficiently addressed issues, and room for bioreactor design improvement and performance optimization. These challenges include creating a triple perfusion bioreactor for vascularized tubular tissue engineered cardiac construct; designing and manufacturing fluidics-based perfused minibioreactors; incorporation of systematic mathematical modeling and computer simulation based on computational fluid dynamics into the bioreactor designing process; and development of automatic systems of hydrodynamic regime control. Designing and engineering of built-in noninvasive biomonitoring systems is another important challenge. The optimal and most efficient perfusion and conditioning regime, which accelerates tissue maturation of tissue-engineered constructs also remains to be determined. This is a first article in a series of reviews on critical elements of cardiovascular tissue engineering technology describing the current status, unsolved problems, and challenges of bioreactor technology in cardiovascular tissue engineering and outlining future trends and developments.