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Evolution of an in vivo bioreactor
Holt, G. E., J. L. Halpern, et al. (2005), J Orthop Res 23(4): 916-23.
Abstract: The ideal bone graft substitute requires osteoconductive, osteoinductive, and osteogenic components. This study introduces an "in vivo bioreactor," a model in which pluripotent cells are recruited from circulating blood to a vascularized coralline scaffold supplemented with bone morphogenetic protein-2 (BMP-2). The bioreactor generates new, ectopic host bone with the capability of vascularized tissue transfer. More importantly, bone is reproducibly formed in a closed and malleable environment. In a rat model, the superficial inferior epigastric vessels were isolated, ligated, and then threaded through a prefabricated coral cylinder (hydroxyapatite, ProOsteon 500). Experimental groups were characterized by the following variables: (1) with/without incorporation of vascular pedicle; (2) with/without addition of BMP-2 (0.02 mg/cm3). Scaffolds were harvested 6 weeks after implantation, embedded and sectioned. Tissue samples were decalcified, fixed, and stained with H&E, trichrome green, and CD31/PECAM-1 (a marker of endothelial cells). Vascularized coral scaffolds supplemented with BMP-2 presumably recruited circulating mesenchymal stem cells to generate bone. Bone formation was quantified through histological analysis, and reported as a percentage, area bone/area cross section scaffold x 100. Mean bone formation was 11.30%+/-1.19. All scaffolds supplied by the vascular pedicle, regardless of BMP-2 supplementation, demonstrated neo-vascular ingrowth. Scaffolds lacking a pedicle showed no evidence of vascular ingrowth or bone formation. This paper introduces a model of a novel "in vivo bioreactor" that has future clinical and research applications. The tissue engineering applications of the "bioreactor" include treatment of skeletal defects (nonunion, tumor post-resection reconstruction). The bioreactor also may serve as a unique model in which to study primary and metastatic cancers of bone.

Evolving concepts in bone tissue engineering
Cowan, C. M., C. Soo, et al. (2005), Curr Top Dev Biol 66: 239-85.
Abstract: The field of tissue engineering integrates the latest advances in molecular biology, biochemistry, engineering, material science, and medical transplantation. Researchers in the developing field of regenerative medicine have identified bone tissue engineering as an attractive translational target. Clinical problems requiring bone regeneration are diverse, and no single regeneration approach will likely resolve all defects. Recent advances in the field of tissue engineering have included the use of sophisticated biocompatible scaffolds, new postnatal multipotent cell populations, and the appropriate cellular stimulation. In particular, synthetic polymer scaffolds allow for fast and reproducible construction, while still retaining biocompatible characteristics. These criteria relate to the immediate goal of determining the ideal implant. The search is becoming a reality with widespread availability of biocompatible scaffolds; however, the desired parameters have not been clearly defined. Currently, most research focuses on the use of bone morphogenetic proteins (BMPs), specifically BMP-2 and BMP-7. These proteins induce osteogenic differentiation in vitro, as well as bone defect healing in vivo. Protein-scaffold interactions that enhance BMP binding are of the utmost importance, since prolonged BMP release creates the most osteogenic microenvironment. Transition into clinical studies has had only mild success and relies on large doses of BMPs for bone formation. Advances within the field of bone tissue engineering will likely overcome these challenges and lead to more clinically relevant therapies.

Ex ova chick chorioallantoic membrane as a novel model for evaluation of tissue responses to biomaterials and implants
Klueh, U., D. I. Dorsky, et al. (2003), J Biomed Mater Res A 67(3): 838-43.
Abstract: One of the major obstacles in developing rationale strategies to control inflammation and fibrosis surrounding implants is the lack of a simple and inexpensive in vivo model to screen tissue reactions to various biomaterials and implants. To begin to fill this gap, we have developed an ex ova model of the chick embryo chorioallantoic membrane (CAM) for testing of tissue reaction to biomaterials and implants. For these studies, we evaluated tissue reactions (inflammation and fibrosis) to two commonly used biomaterials (nylon and silastic) grossly and histologically in the ex ova CAM. Nylon mesh was incorporated within the CAM tissue 4 days postplacement. After 8 days postplacement, the nylon mesh was totally incorporated into the CAM. Histologically, little or no inflammation was seen associated with the incorporated nylon mesh at any time point. In the case of silastic tubing, significant incorporation of the CAM was seen grossly by 1-2 days postplacement. Incorporation of the tubing continued at day 8 postplacement of the silastic tubing, with ingrowth of the CAM into the lumen of the tubing. Histological evaluation of CAMs indicated that no significant tissue reactions (inflammation or fibrosis) occurred in the CAM tissue surrounding the silastic tubing or in the CAM tissue and vasculature that had grown into the silastic tubing. To our knowledge, this report represents the first investigation of the usage of the ex ova CAM model, a shell-less chick embryo model (ex ova), as an in vivo model to test the tissue reactions to biomaterials and implants.

EXAFS and XANES studies of retention of copper and lead by a lignocellulosic biomaterial
Dupont, L., E. Guillon, et al. (2002), Environ Sci Technol 36(23): 5062-6.
Abstract: Lignocellulosic substrate (LS), which is a low cost biomaterial, has a strong complexing ability and can be used in the treatment of wastewaters as biosorbentto remove heavy metals. The speciation of copper and lead to this biomaterial has been studied by X-ray absorption spectroscopy. The copper(II) has a 6-coordinate structure with four oxygen atoms in the equatorial plane at 1.95 A and two in axial position at 2.35 A. In the case of lead a particularly low coordination number of about 3 has been obtained. The combination of extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structure (XANES) suggested that Cu and Pb are bound to the surface of LS through carboxylic moieties.

Examination of membrane rupture as a mechanism for mammalian cell detachment from fibronectin-coated biomaterials
Goldstein, A. S. and P. A. DiMilla (2003), J Biomed Mater Res A 67(2): 658-66.
Abstract: Synthetic biomaterials intended for the reconstruction of tissues and organs must be capable of sustaining adhesive contact with adjacent cells and tissues under mechanical and hydrodynamic stresses. To facilitate this adhesion, extracellular matrix proteins or peptide sequences are frequently immobilized to the biomaterial interface. These ligands enhance cell attachment by raising the number of cell receptor/ligand interactions, but consequently they may alter the mechanism of cell detachment. In particular, as the cell membrane is more strongly immobilized to the substratum, the tendency for cell detachment to involve membrane rupture may increase. To test this hypothesis, cells were fluorescent stained with a membrane dye, allowed to attach to fibronectin-coated model substrates for 30 min, and then subjected to a spatially dependent range of shear stress for 5 min (28-220 dyn/cm2) using a radial-flow chamber. Phase-contrast and fluorescent images were analyzed to determine the probability for cell detachment and the area of fluorescent debris left by detaching cells as a function of fibronectin concentration, magnitude of shear stress, and time. It was found at all concentrations of fibronectin that the majority of detaching cells left membrane fragments, the mean size of these fragments was independent of shear stress, and the shape independent of the direction of flow. However, mean fragment area increased with concentration of fibronectin and decreased with duration of shearing flow. We postulate that the area of debris reflects the extent of cell attachment prior to the application of shear and that adhesive complexes can disassemble at the onset of flow.

Examination of osteoblast-orthopaedic biomaterial interactions using molecular techniques
Puleo, D. A., K. E. Preston, et al. (1993), Biomaterials 14(2): 111-4.
Abstract: Molecular techniques can be used to elucidate the effects of extended periods of cell-biomaterial interactions on the time-course and level of expression of particular genes which determine cellular phenotype. We used the polymerase chain reaction to demonstrate the expression of genes for the bone-related proteins osteocalcin, osteonectin and osteopontin by neonatal rat calvarial osteoblasts. In addition, Northern blotting was subsequently used to show that messenger RNAs encoding osteonectin and osteopontin were consistently expressed during a 5 wk period of interaction of osteoblasts with Ti-6Al-4V, a commercial brand of hydroxyapatite, and tissue culture polystyrene.

Excellent results from proximally HA-coated femoral stems with a minimum of 6 years follow-up: a prospective evaluation of 100 patients
Goosen, J. H., A. J. Swieringa, et al. (2005), Acta Orthop 76(2): 190-7.
Abstract: BACKGROUND: There have been few reports on the mid- and long-term follow-up results of the proximally HA-coated femoral stem. We evaluated this type of stem prospectively, with 6-12 years of follow-up. METHODS: The survival rate, Harris hip score and radiographic features of 106 hips in 100 consecutive patients were evaluated. The mean age at operation was 51 years (SD 8.2). RESULTS: The mean Harris hip score at the time of the latest follow-up was 95 points. Spot welds occurred in 95% of the patients and were first observed at a mean follow-up of 1.4 years in one or more of the Gruen regions, corresponding to the coated part of the femoral stem. A higher grade of stress shielding correlated with a less favorable Harris hip score and pain subscore. According to the criteria of Engh, all stems were graded as stable and durable bone-ingrown. No femoral component was revised. INTERPRETATION: At an average follow-up of 8 years, this proximally HA-coated femoral component showed favorable clinical and radiological outcome and excellent survivorship.

Excretion of resorption products from bioactive glass implanted in rabbit muscle
Lai, W., J. Garino, et al. (2005), J Biomed Mater Res A 75(2): 398-407.
Abstract: Bioactive glass granules were implanted in the paraspinal muscle of rabbits to determine the pathway of the silicon released from bioactive glass in vivo. We traced and quantified the silicon released by obtaining 24-h urine and blood samples for up to 6 months after implantation. Furthermore, local muscle tissue as well as the following organs were resected for chemical and histopathological analyses: brain, heart, kidney, liver, lung, lymph nodes, spleen, and thymus. The urinary silicon of the group with implanted granules was significantly higher than in the sham-operated, control group. The average excretion rate was 2.4 mg/day, and as such, 100% of the implanted silicon was excreted in 19 weeks. No elevated concentrations of silicon were found at the implant sites or in the other organs at sacrifice, that is, 24 weeks. The histological appearance of all organs was normal throughout. The concentrations of silicon measured in the urine were well below saturation and because no significant increase in silicon was found in any organ, including kidney, the increased silicon excretion rate was within the physiological capacity of rabbits. Therefore, it can be concluded that the resorbed silica gel is harmlessly excreted in soluble form through the urine.

Expansion anchors for use in anterior cruciate ligament (ACL) reconstruction: establishing proof of concept in a benchtop analysis
Black, K. P. and M. M. Saunders (2005), Med Eng Phys 27(5): 425-34.
Abstract: The current method for graft fixation in bone tendon-bone anterior cruciate ligament (ACL) reconstruction is the interference screw. Although this method of fixation provides for adequate graft fixation with respect to strength, intraoperative placement is difficult and the failure rate is high. To address these concerns, we have designed and fabricated prototype expansion anchors that could be expanded to anchor the graft in the bone tunnel. As a first step in assessing the validity of this concept, in the current work, we demonstrate that these systems are of comparable fixation strength (biomechanical pullout testing) to the standard interference screw, are smaller at the time of insertion and thus provide for increased visibility and ease of placement. The increased visibility should result in better placement and reduced failure rates. The increased ease of placement should result in significant savings in decreased OR time.

Expansion of human chondrocytes in an intermittent stirred flow bioreactor, using modified biodegradable microspheres
Curran, S. J., R. Chen, et al. (2005), Tissue Eng 11(9-10): 1312-22.

Experience with management of anterior abdominal wall defects using bovine pericard
Tuil, C. V., A. K. Saxena, et al. (2005), Hernia: 1-7.
Abstract: During 5 years from 1999 until 2003, our experience with 29 (100%) neonates managed for anterior abdominal wall defects is presented. Twenty-one (72%) neonates presented with gastroschisis and 8 (28%) neonates with giant omphaloceles. The male:female ratio was almost equal in gastroschisis (1:1) while a male predominance was observed in omphaloceles (6:1). A primary closure of the defect was possible in 5 (17%) cases and a single patch along with skin closure was achieved in a further 9 (31%) cases. In 15 (52%) neonates the defect was large and two patches were employed to sufficiently cover the defect. All patients (97%), except one (mortality due to extreme prematurity), were managed successfully. Depending upon the size of the defect and the metabolic condition of the neonate, the defect closure was completed after a mean of 85.7 days. Special protocols were created to manage the bovine pericard patches, which behaved differently to lyophilized dura patches previously used at our center. Integration of the patches was successful in 28 (97%) neonates; however, one neonate with gastroschisis presented significant challenges in the management. Bovine pericard patches are optimal biomaterials for the closure of anterior abdominal wall defects in gastroschisis and omphaloceles.

Experience with osseous implants for bone and biomaterials research
Fox, W. C. and T. B. Aufdemorte (1993), J Long Term Eff Med Implants 3(1): 1-27.
Abstract: A Cancellous Access Port (CAP) osseous implant for repeated sampling of cancellous tissue was developed and tested for experimental studies of bone, marrow, and materials biocompatibility. The CAP and associated methods were shown in a series of studies to allow the repeated biopsy of unstrained cancellous tissue with little morbidity and no mortality when used in baboons, rhesus, sheep, and horses. CAP infusion, material testing, and biopsy core assemblies were developed and tested for the administration and assessment of the effects of pharmaceuticals and biologics; the placement and retrieval of a biomaterial with the surrounding tissue; and studies of cancellous bone healing and remodeling.

Experimental approaches to study vascularization in tissue engineering and biomaterial applications
Kirkpatrick, C. J., R. E. Unger, et al. (2003), J Mater Sci Mater Med 14(8): 677-81.
Abstract: The success of tissue engineering and biomaterial applications is not only dependent on the growth and functioning of the organ- or tissue-specific cells on the biomaterial but is entirely dependent in most cases on a successful vascularization after implantation. The process of vascularization involves angiogenesis; the formation of new blood vessels which spread into the implant material and supply the existing cells with the nutrients to survive. We have established in vitro methods using human microvascular endothelial cells to evaluate novel biomaterials for endothelial cell attachment, cytotoxicity, growth, angiogenesis and the effects on gene regulation. These in vitro studies can be used to rapidly evaluate the potential success of a new biomaterial and for the development of matrix scaffolds which will promote a physiological vascularization response.

Experimental evaluation of a percutaneous injectable biomaterial used in radio-interventional bone-filling procedures
Pasquier, G., B. Flautre, et al. (1998), J Mater Sci Mater Med 9(6): 333-6.
Abstract: Interventional radiology is beginning to be used in bone pathology. An injectable biomaterial Ethibloc*, composed of alcohol and a vegetal protein (zein), has been successfully used for percutaneous treatment of benign bone lesions. The reasons for this success remained unknown and needed to be understood. In this study, using a rabbit model, an evaluation was made of bone formation and tissue reaction during the first weeks after the injection of this biomaterial. Ethibloc* was injected percutaneously into bone defects in rabbit distal femurs. Three time intervals were studied: 2, 4 and 8 wks. The three control groups constituted unfilled, polymethylmethacrylate (PMMA), and alcohol. Undecalcified bone technique was used for a qualitative analysis and histomorphometric evaluation. A low bone formation was found which was less than in the control groups (PMMA and unfilled). The "Ethibloc group" was characterized by an early inflammatory reaction. Good clinical results obtained with Ethibloc* probably arose, after an initial vascular thrombosis, from a secondary bone reaction and spontaneous osteogenesis obtained after the disappearance of vascular or hyperpression reaction.

Experimental evaluation of three biomaterials for replacement of the pulmonary artery branches
Planche, C., E. Onursal, et al. (1983), Eur Surg Res 15(2): 79-91.
Abstract: 24 adult dogs were randomly divided into 4 groups and submitted to angioplastic replacement of a 2- to 3-cm segment of the right or left pulmonary artery by three kinds of autogenous biomaterials; azygous vein, jugular vein and pericardium, and by a synthetic arterial prosthesis in a control group. In vivo evaluation was carried out by catheterization with a balloon catheter allowing successive intraluminal occlusion of the pulmonary branches, and by cineangiopneumography. There was a good correlation between evaluation techniques. Final evaluation after a follow-up of 5-20 months was based on postmortem measurement of the anastomosis diameter and histologic examination. In each group, the results were classified into four grades, numbered 0-3, and compared statistically. The results show that in this experimental model: (a) none of the autogenous tissues (selected for their potential ability to grow) proved better than the control Dacron arterial prosthesis, and (b) of the three biomaterials, the azygous vein was superior to the jugular vein and pericardium.

Experimental implantation and long-term testing of an intraocular vision aid in rabbits
Szurman, P., M. Warga, et al. (2005), Arch Ophthalmol 123(7): 964-9.
Abstract: OBJECTIVE: To develop an intraocular vision aid to provide artificial vision in severely traumatized eyes, where neuroretinal function could be preserved but irreversible anterior segment opacification resulted in blindness. METHODS: The basis of an intraocular vision aid is in principle a telemetric circuit to bridge the opaque cornea and to allow for artificial light stimulation of the retina. The visual prosthesis comprises an external high-dynamic range complementary metal oxide semiconductor camera and digital signal processing unit and an intraocular miniaturized light-emitting diode array to project the image onto the retina. For in vivo testing of long-term function and biocompatibility, silicone-encapsulated active photodiodes were implanted in 13 pigmented rabbits and were followed up for up to 21 months. RESULTS: Lens extraction and stable fixation of the device in the ciliary sulcus were successful in all cases. For up to 21 months inductive energy transmission and wireless stimulation of the implants could be maintained. Electrophysiologic data and histology demonstrated a good tissue biocompatibility in the long-term follow-up. CONCLUSION: The results demonstrate the general feasibility and biocompatibility to implant and fixate an intraocular light-emitting diode prosthesis. Inductive energy transmission to the intraocular device and wireless light stimulation are assured in the long term but depend on meticulous water-impermeable encapsulation of the delicate microelectronic components.Clinical Relevance An intraocular vision aid compound system with a high-resolution light-emitting diode matrix might be a future treatment option to restore vision in blind eyes with severe anterior segment disorders.

Experimental in vitro and in vivo studies of epithelium formation on biomaterials seeded with isolated respiratory cells
Kaschke, O., H. J. Gerhardt, et al. (1996), J Invest Surg 9(2): 59-79.
Abstract: Extensive tracheal defects after intensive care medicine, trauma, or large resections in tumor surgery remain a major challenge in plastic and reconstructive surgery. Defects that cannot be satisfactorily treated by complicated and costly reconstructive techniques reveal a need for an alloplastic tracheal replacement. Recent experimental and clinical studies in the development of alloplastic tracheal prostheses proved that the lack of an epithelial lining on the luminal surfaces and inadequate biophysical properties and shapes of the prostheses were the main causes for failure of these prostheses. In this study a cell-seeding technique has been used. Adhesion, spreading, and differentiation of seeded mucosa cells on biomaterials in vitro were observed by scanning electron microscopy (SEM). Chemical properties and surface structure of the material influenced the differentiation process. Epithelium formation of incorporated tracheal prostheses was tested in animal experiments. Isolated respiratory cells were seeded into implanted tubular prostheses of porous polyurethane or expanded polytetrafluorethylene. Light microscopy and SEM showed the tendency of epithelium formation on the surface of the lumen. Vigorous cell layers, predominantly as multiple cell layers of squamous epithelium, were observed. Ciliated or mucus cells were not detected. It can be stated that the epithelium formation on incorporated porous implants is possible. Further studies of the stability and the differentiation process of the epithelium on such implants is needed before an introduction of tracheal replacements into the clinical practice can be considered.

Experimental study of an adjustable-length prosthesis in a temporal bone model
Zhao, S., N. Hato, et al. (2005), Acta Otolaryngol 125(1): 33-7.
Abstract: CONCLUSIONS: This prosthesis has the advantage of rapid adjustment at the time of insertion in order to achieve optimal tension and, as a result, optimal sound transmission. OBJECTIVE: To test the acoustic performance of a new, adjustable incus replacement prosthesis in a human temporal bone model. MATERIAL AND METHODS: Experiments were performed in seven human temporal bones, before and after removal of the incus and insertion of the prosthesis. The input comprised 406 pure tones ranging in frequency between 0.1 and 10 kHz at an intensity of 80 dB SPL at the tympanic membrane. The output measurement was stapes footplate displacement, determined by means of a laser Doppler vibrometer. Three lengths of the prosthesis were investigated: optimal, optimal +0.2 mm and optimal +0.4 mm. RESULTS: The optimal-length prosthesis produced similar results to those of an intact middle ear. The slightly longer prostheses decreased middle ear sound transmission at all test frequencies, except those near 1.5 kHz.

Experimental study of bone formation around a titanium rod with beta-tricalcium phosphate and prostaglandin E2 receptor agonists
Masuzawa, M., M. Beppu, et al. (2005), J Orthop Sci 10(3): 308-14.
Abstract: beta-Tricalcium phosphate (beta-TCP) is an excellent bone-filling material that is completely absorbed by the body and replaced by autologous bone. Unfortunately, its mechanical strength is low, rendering its application at loaded regions difficult. The purpose of this study is to evaluate the histological and mechanical effects of single and combined use of beta-TCP and EP4 agonist on bone formation around a titanium rod. beta-TCP was loaded into the femoral bone marrow from the distal end of the femur, where the titanium implants were inserted, and the animals received twice-daily subcutaneous injections of EP4 agonist. Group I received the rod only and was designated the control group; group II received EP4 agonist only; group III received beta-TCP only; and group IV received both beta-TCP and EP4 agonist. Examination of decalcified specimens revealed favorable bone formation in all treatment groups compared with that in group I, with the most active bone formation seen in group IV. Mechanical evaluation revealed significant differences in maximum pull-out force compared with group I at weeks 4 and 8. There were no differences between groups II and III at either week 4 or 8, but the values seen in group IV at weeks 4 and 8 were significantly higher compared with the other groups. Combined use of beta-TCP and EP4 agonist is expected to compensate for bone defects resulting from revision total joint arthroplasty and to achieve stability at an early stage.

Exploring and engineering the cell surface interface
Stevens, M. M. and J. H. George (2005), Science 310(5751): 1135-8.
Abstract: Cells are inherently sensitive to local mesoscale, microscale, and nanoscale patterns of chemistry and topography. We review current approaches to control cell behavior through the nanoscale engineering of materials surfaces. Far-reaching implications are emerging for applications including medical implants, cell supports, and materials that can be used as instructive three-dimensional environments for tissue regeneration.

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Last Modified: 8 February 2006