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Tissue and bone engineering are essential components for understanding of the healing and regeneration of bone and adjacent tissues. Recently, interest in restoring the contour and shape in the configuration of the reconstructed bone has become a priority for craniofacial surgeons. This article discusses the latest advancements in bone tissue engineering applications.
In the context of minimally invasive surgery, the next logical step is to provide a biological replacement for missing tissue without the need for a harvesting operation. Tissue engineering is defined as the fabrication of living parts for the body from cells in the laboratory. Donor cells such as stem cell or cultivated, differentiated cells are seeded on a appropriately configurated scaffold replicating extracellular matrix. Growth factors are added to the in-vitro system to encourage stem cell proliferation. The engineered structure is then transplanted to the recipient. In this article, the regeneration strategies for tooth and periodontal tissue using tissue engineering concepts. Culture expanded mesenchymal stem cell is useful for periodontal tissue and tooth regeneration with bioabsorbable matrix such as b-TCP and collagen fiber. In the near future, these regenerated tissue will be accepted in clinical situations.
Recently developed perfusion micro-bioreactors offer the promise of more physiologic in vitro systems for tissue engineering. Successful application of such bioreactors will require a method to characterize the bioreactor environment required to elicit desired cell function. We present a mathematical model to describe nutrient/growth factor transport and cell growth inside a microchannel bioreactor. Using the model, we first show that the nature of spatial gradients in nutrient concentration can be controlled by both design and operating conditions and are a strong function of cell uptake rates. Next, we extend our model to investigate the spatial distributions of cell-secreted soluble autocrine/paracrine growth factors in the bioreactor. We show that the convective transport associated with the continuous cell culture and possible media recirculation can significantly alter the concentration distribution of the soluble signaling molecules as compared to static culture experiments and hence needs special attention when adapting static culture protocols for the bioreactor. Further, using an unsteady state model, we find that spatial gradients in nutrient/growth factor concentrations can bring about spatial variations in the cell density distribution inside the bioreactor, which can result in lowered working volume of the bioreactor. Finally, we show that the nutrient and spatial limitations can dramatically affect the composition of a co-cultured cell population. Our results are significant for the development, design, and optimization of novel micro-channel systems for tissue engineering.
BACKGROUND: The standard therapy for short bowel syndrome is total parenteral nutrition, which is expensive and associated with significant morbidity and mortality. New therapeutic approaches for this disorder are needed. We have applied the techniques of tissue engineering to develop a prototype neointestine. We hypothesized that anastomosis of this neointestine to the native bowel would result in regeneration of mucosal morphology and enterocyte dynamics. METHODS: Biodegradable polymers seeded with neonatal rat intestinal organoid units were implanted into the omenta of adult rats to form neointestinal cysts. Five weeks after implantation, side-to-side cyst-jejunal anastomoses were fashioned in one cohort of rats. Tissues were harvested from all rats at 5 months after implantation. Native jejunal (J) and non-anastomosed (N-N) and anastomosed (A-N) neointestinal tissues were assessed for morphology, epithelial cell proliferation (5-bromo-2-deoxyuridine immunohistochemistry), apoptotic rates (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay), and SGLT1 in situ hybridization. RESULTS: Mucosal morphology, rates and topography of enterocyte proliferation, and transporter expression in A-N neointestine recapitulated those of native jejunum. Each of these features was rudimentary in N-N neointestine. CONCLUSIONS: These results suggest that the tissue-engineered neomucosa can develop structural and dynamic features of the normal jejunum. Anastomosis to the native intestine is an essential step for neomucosal development. Tissue engineering offers promise as a novel approach to the treatment of patients suffering from short bowel syndrome.
OBJECTIVE: The aim of this study was to compare bone regeneration after grafting enucleated mandibular cyst cavities using either autogenous osteoblasts cultured on a biomaterial or autogenous spongiose iliac bone. STUDY DESIGN: Twenty patients with 22 mandibular cysts were assessed. Eleven cysts were filled in with tissue-engineered bone (autogenous osteblasts cultured on demineralized bone matrix Osteovit) and 11 with spongiose iliac bone as controls. Panoramic radiographs were taken preoperatively, immediately postoperatively, and 3, 6, and 12 months after surgery. Radiolucency was computer analyzed using gray-level histograms. RESULTS: In both groups bone regeneration took place in a similar fashion. After 3 and 6 months there were few differences in bone density between the groups. However, in radiographic controls after 12 months ossification was considerably stronger in cysts grafted with tissue-engineered bone. CONCLUSION: These results advocate for the clinical application of tissue-engineered bone as an alternative viable filling material for cysts.
PURPOSE: This article describes the use of tissue-engineered mucosal grafts instead of split-thickness skin grafts after freeing of the tongue in patients who had previous resection of an oral squamous cell carcinoma and initial primary wound closure. PATIENTS AND METHODS: Tissue-engineered mucosal grafts, up to 75 cm2 in size, were cultured from biopsy specimens of the hard palate in 6 patients, starting 3 to 4 weeks before the operation. After freeing of the tongue, the engineered mucosa was implanted on the wound surface by using vaseline gauze as carrier and fixed with an intraoral gauze wound dressing. RESULTS: A good glossoalveolar sulcus was formed in 5 patients, resulting in good mobility of the tongue and a satisfactory denture-bearing surface. In 1 patient, there was a disturbance of wound healing, leading to severe shrinkage of the glossoalveolar sulcus and very limited improvement in tongue mobility. Preoperative bromodeoxyuridine (BrdU) labeling of the graft and postoperative immunohistochemical staining of biopsy specimens from the grafted areas with anti-BrdU showed that the cultured cells are integrated into the newly formed mucosal epithelium. Postoperative histologic investigations showed a differentiation process in the grafted mucosal epithelium, with a change in the expression of cytokeratins. At 6 months postoperatively, the typical pattern of normal nongrafted mucosa was regained. CONCLUSIONS: This investigation provides evidence that tissue-engineered mucosal cells can serve as a graft for large intraoral wounds. Complete intraoral lining is quickly reestablished, and normal epithelial differentiation is seen in the graft area within a 6-month postoperative period.
PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.
OBJECTIVE: The purpose of this study was to evaluate the feasibility of using autologous sheep marrow stromal cells cultured onto polyglycolic acid mesh to develop helical engineered cartilage equivalents for a functional tracheal replacement. We also explored the potential benefit of local delivery of transforming growth factor beta 2 with biodegradable gelatin microspheres. METHODS: Bone marrow was obtained by iliac crest aspiration from 6-month-old sheep and cultured in monolayer for 2 weeks. At confluence, the cells were seeded onto nonwoven polyglycolic acid fiber mesh and cultured in vitro with transforming growth factor beta 2 and insulin-like growth factor 1 for 1 week. Cell-polymer constructs were wrapped around a silicone helical template. Constructs were then coated with microspheres incorporating 0.5 microg transforming growth factor beta 2. The cell-polymer-microsphere structures were then implanted into a nude rat. On removal, glycosaminoglycan content and hydroxyproline were analyzed in both native and tissue-engineered trachea. Histologic sections of both native and tissue-engineered trachea were stained with hematoxylin and eosin, safranin-O, and a monoclonal anti-type II collagen antibody. RESULTS: Cell-polymer constructs with transforming growth factor beta 2 microspheres formed stiff cartilage de novo in the shape of a helix after 6 weeks. Control constructs lacking transforming growth factor beta 2 microspheres appeared to be much stiffer than typical cartilage, with an apparently mineralized matrix. Tissue-engineered trachea was similar to normal trachea. Histologic data showed the presence of mature cartilage. Glycosaminoglycan and hydroxyproline contents were also similar to native cartilage levels. CONCLUSIONS: This study demonstrates the feasibility of engineering tracheas with sheep marrow stromal cells as a cell source. Engineering the tracheal equivalents with supplemental transforming growth factor beta 2 seemed to have a positive effect on retaining a cartilaginous phenotype in the newly forming tissue.
PURPOSE: We previously reported a method for the development of tissue-engineered tooth. However, 1 drawback of the procedure was the inability to determine whether the tooth would function when transplanted in the jaw because it was formed in the omentum of the abdomen. Therefore, the present study was designed to evaluate whether transplantation of dissociated odontogenic cells could induce tissue-engineered odontogenesis in the canine jaw. MATERIALS AND METHODS: Cells were harvested from canine first molar tooth buds and the resulting heterogeneous cell population was seeded on a biodegradable polymer. These constructs were then transplanted into the same sockets after extracting the tooth buds. After transplantation, we evaluated the transplanted constructs using dental x-ray, micro-computed tomography, histology, and immunohistochemistry. RESULTS: After 24 weeks, micro-x-ray computed tomography showed regenerated hard tissues in the jaw, and hematoxylin and eosin staining showed tubular dentin and bone. In the regenerated tissue, osteopontin, osteonectin, and osteocalcin antibodies stained the dentinal matrix. However, enamel tissue and dental-root formation were not observed. CONCLUSION: These data show for the first time the formation of dentin and bone from dissociated odontogenic cells in the canine jaw.
PURPOSE: We have previously shown that fetal tissue engineering is a preferred alternative to diaphragmatic repair in a large animal model. This study was aimed at comparing diaphragmatic constructs seeded with mesenchymal amniocytes and fetal myoblasts in this model. METHODS: Neonatal lambs (n = 14) underwent repair of an experimental diaphragmatic defect with identical scaffolds, either seeded with labeled autologous cells (mesenchymal amniocytes in group 1 and fetal myoblasts in group 2) or as an acellular graft (group 3). At 1 to 12 months postoperatively, implants were harvested for multiple analyses. RESULTS: Repair failure (reherniation or eventration) was significantly higher in group 3 than in groups 1 and 2, with no difference between groups 1 and 2. Seeded fetal myoblasts quickly lost their myogenic phenotype in vivo. All grafts contained cells with a fibroblastic-myofibroblastic profile. Elastin concentrations and both modular and ultimate tensile strengths were significantly higher in group 1 than in groups 2 and 3. There were no differences in glycosaminoglycans and type I collagen levels among the groups. CONCLUSIONS: Diaphragmatic repair with a mesenchymal amniocyte-based engineered tendon leads to improved structural outcomes when compared with equivalent fetal myoblast-based and acellular grafts. The amniotic fluid is a preferred cell source for tissue-engineered diaphragmatic reconstruction.
Current progress integrating stem cell biology and tissue engineering techniques has been invaluable to clinical applications. Prior to the application of cellular transplantation technique to patients, we need to establish techniques that can monitor their tissue biodistribution non-invasively. In this study, we proposed an imaging modality using MRI to not only monitor implanted scaffold in vivo, but also to track transplanted cells and behavior around the implant. For this purpose, human bone marrow-derived mesenchymal stem cells (hMSCs) were labeled with superparamagnetic iron oxide (Feridex) and then labeled hMSCs were cultured in a gelatin sponge used as a scaffold to support cell growth and proliferation. Histological assessment and MTT assay showed that cell labeling with MR contrast agent did not harm cell viability. Also, Feridex-labeled hMSCs showed a significant decrease in T2 signal intensity, even within the gelatin sponge in vitro. After implanting the sponge/cell complex in vivo, we could visualize cellular behavior around the implant over time using a noninvasive MRI modality and this finding was correlated with histological study, which illustrates the potential of a new approach proposed here for in vivo monitoring of implanted cell-based tissue-engineered product.
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Patient own fibrin may act as the safest, cheapest and immediate available biodegradable scaffold material in clinical 1 tissue engineering. This study investigated the feasibility of using patient own fibrin isolated from whole blood to construct a new human cartilage, skin and bone. Constructed in vitro tissues were implanted on the dorsal part of the nude mice for in vivo maturation. After 8 weeks of implantation, the engineered tissues were removed for histological analysis. Our results demonstrated autologous fibrin has great potential as clinical scaffold material to construct various human tissues.
OBJECTIVE: To summarize the latest developments in silk protein fiber as biomaterials and their applications in tissue engineering. METHODS: Recent original literature on silk protein fiber as biomaterials were reviewed, illustrating the properties of silk protein fiber biomaterials. RESULTS: The silk protein fiber has the same functions of supporting the cell adhesion, differentiation and growth as native collagen, and is renewed as novel biomaterials with good biocompatibility, unique mechanical properties and is degradable over a longer time. CONCLUSION: Silk protein fiber can be used as a suitable matrix for three dimensional cell culture in tissue engineering. It has a great potential applications in other fields.
Cell therapy and tissue engineering may well likely dominate medical therapeutics in the next century. Growing a functional glomerular filter and tubule reabsorber from a combination of cells, biomaterials, and synthetic polymers to replace renal excretory and regulatory functions is a specific example of these evolving technologies. The kidney was the first organ whose function was substituted by an artificial device. The kidney was also the first organ to be successfully transplanted. The ability to replace renal function with these revolutionary technologies in the past was due to the fact that renal excretory function is based on natural physical forces which govern solute and fluid movement from the body compartment to the external environment. The need for coordinated mechanical or electrical activities got renal substitution was not required. Accordingly, the kidney may well be the first organ to be available as a tissue-engineered implantable device as a fully functional replacement part for the human body. The prospects of a "universal donor" bioartificial kidney for the treatment of end-stage renal disease are clearly achievable as we approach the next millennium.
OBJECTIVE: To investigate the preparation method of porcine bladder acellular matrix graft (BAMG) and evaluate the feasibility of using BAMG as biomaterial scaffold to construct tissue engineering bladder. METHODS: Bladders were harvested from adult pigs at a slaughterhouse and were decellularized by the method of enzyme digestion or eradicator washing. The BAMG was then examined by optic microscope and electron microscope to confirm no cell elements remained. The structure of the BAMG was always observed through the Scanning Microscope. The skeletal muscle stem cells of New Zealand white rabbits were implanted in 96-hole-plank and cultured by BAMG extract liquid or normal culture medium. The cytotoxicity of BAMG was tested through MTT assay. New Zealand white rabbits were underwent bladder triangle area spared cystectomy and reconstructed by the heterogeneity BAMG. The animal bladders were obtained after six weeks and the tissue regeneration was examined. RESULTS: Good outcome were obtained with either of the two methods. There were no cell elements remained under the examination of optical microscope and electron microscope. The interstice could observed on BAMG through the Scanning Microscope. The relative growth rate (RGR) was 81%, 88%, 93% and 96% separately on the 2nd, the 4th, the 6th and the 8th day (t = 3.7419, P < 0.025). The cytotoxicity score was I, which indicated that BAMG had a good cytocompatibility. All rabbits were alive except one which died from diarrhea. The reconstructed bladder resumed the construction and function of the bladder. The transitional cells and muscle cells growth could be seen in the scaffold of the repaired bladder after six weeks. CONCLUSION: The porcine BAMG has good biocompatibility and can be used as ideal biomaterial scaffold of bladder tissue engineering.
Reliable and reproducible outcome measures are essential to assess the efficacy of competing and novel tissue-engineering techniques. The aim of this study was to compare traditional histological analyses with newly developed quantitative biochemical outcome measures for the repair of articular cartilage. The production of a new anti-peptide antibody and the development and validation of a novel method for the extraction and immunoassay of type I collagen are described. The assay was used, in conjunction with existing assays for type II collagen and proteoglycans, to measure levels of the matrix components in repair tissue biopsies obtained from patients treated with the new tissue-engineering therapy Hyalograft C. Frozen sections cut from the same biopsies were stained for proteoglycans, using safranin O, and immunohistochemical analysis was used to assess type I and II collagen staining. Although there was general agreement between the extent of staining and the amounts of the three matrix components, there was a large degree of overlap in biochemical content between biopsies classified histologically on the basis of low, moderate, or abundant staining. The results demonstrate that histological grading of matrix protein abundance to classify repair cartilage as hyaline or fibrocartilagenous is often misleading. In addition, we demonstrate for the first time the ability to measure collagen cross-links in repair tissue biopsies and show that it can be used as a surrogate marker for tissue maturity. Our new range of biochemical techniques provides a standardized method to assess the quality of both engineered cartilage produced in vitro and repair tissue biopsies obtained after in vivo implantation.
OBJECTIVE: To look for the best carrier for cultivating oral tissue engineered mucosa. METHODS: A series of membrane of scaffold materials of polycleclide-co-plycoclide (PLGA) were studied on their weight and biocompatibility after they had been implanted subcutaneously in rabbits for 1, 2, 3, 4 weeks respectively. RESULTS: PLGA I , II, III degraded completely in rabbits after 2, 3, 4 weeks respectively. The other PLGA membrane degraded about 50% after 4 weeks. Histologically, the reactions of PLGA I, II, III with surrounding tissues were normal and membranes had a good biocompatibility. CONCLUSION: The biodegrading rate of PLGA II is suitable for clinic practice. PLGA II was a promising carrier for oral tissue-engineered mucosa due to its excellent biocompatibility and biodegrading rate.