Assessments of injectable alginate particle-embedded fibrin hydrogels for soft tissue reconstruction.

Soft tissue reconstruction is often needed after massive traumatic damage or cancer removal. In this study, we developed a novel hybrid hydrogel system consisting of alginate particles and a fibrin matrix that could maintain tissue volume long term. Alginate particles were fabricated by mixing 5% alginate with a 20 mM calcium solution. Cells and these alginate particles were then embedded in fibrin (alginate-fibrin) hydrogels using a dual syringe mixer. Cell-hydrogel constructs were evaluated in terms of cell survival and proliferation in the constructs in vitro. The results indicated that cellular viability, spreading and proliferation in the alginate-fibrin hydrogels were significantly higher compared to constructs fabricated with fibrin or alginate only. In vivo explants showed that cells contained within fibrin-only hydrogels did not contribute to neo-tissue formation, and the fibrin was fully degraded within a 12 week period. In the alginate-fibrin system, higher cellularity and vascular ingrowth were observed in vivo. This resulted in neo-tissue formation in the alginate-fibrin hydrogels. These results demonstrate that fibrin may enhance cell proliferation and accelerate the formation of extracellular matrix proteins in the alginate-fibrin system, while the alginate particles could contribute to volume retention. This injectable hybrid system composed of degradable and non-degradable hydrogels may be a preferable approach to the repair of soft tissue defects.

Adipose stem cells for soft tissue regeneration.

Adipose-derived stem cells (ASCs) can be isolated from human adipose tissue with the exceptional potential for differentiation into mature adipocytes. Utilization of this system is very promising in developing improved techniques to repair soft tissue defects. Current reconstructive procedures, especially after trauma and oncological surgery, transfer autologous soft tissue grafts having limitations. However, ASCs offer the ability to either generate soft tissue with no donor-site morbidity (with the exception of a minor loss of adipose tissue) or enhance the viability and durability of other grafts. This review will discuss the relevant properties of human adult adipose-derived stem cells for the regeneration of adipose tissue. Discussion will focus on the biology of ASCs, cell delivery vehicles/scaffolds useful in applying ASCs as a therapy, and suitable IN VIVO animal models for studying adipose tissue engineering. Also included is a description of the current clinical studies with ASCs in Europe and Asia.

Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation.

Cells and tissues in vivo are subjected to various forms of mechanical forces that are essential to their normal development and functions. The arterial blood vessel wall is continuously exposed to mechanical stresses such as pressure, strain, and shear due to the pulsatile nature of blood flow. Vascular smooth muscle cells (SMCs) populate the media of blood vessels and play important roles in the control of vasoactivity and the remodeling of the vessel wall. It is well documented that the phenotype and functions of vascular SMCs are not only regulated by chemical factors such as transforming growth factor-beta(1) (TGF-beta(1)), but also by mechanical factors such as uniaxial strain. The purpose of our study was to explore the effects of TGF-beta(1) alone or in combination with uniaxial cyclic strain on adipose-derived stem cell (ASC) morphology, proliferation, and differentiation. Low passage ASCs were stimulated with 10% strain at 1 Hz for 7 days, with or without TGF-beta(1). Cyclic strain inhibited proliferation, and caused alignment of the cells and of the F-actin cytoskeleton perpendicular to the direction of strain. Strain alone resulted in a decrease in the expression of early SMC markers alpha-SMA and h (1)-calponin. While the response of SMCs and other progenitor cells such as bone marrow stromal cells to mechanical forces has been extensively studied, the roles of these forces on ASCs remain unexplored. This work advances our understanding of the mechanical regulation of ASCs.

Tolerance to limb tissue allografts between swine matched for major histocompatibility complex antigens.

Transplantation of limb tissue allografts would greatly expand the realm of reconstructive surgery. However, the toxicity of chronic immunosuppression has adversely tilted the risk-benefit balance for clinical transplant. In this study, a procedure was sought to achieve host tolerance to limb tissue allografts through matching of the major histocompatibility complex (MHC) antigens between donor and host swine using only a 12-day course of cyclosporine. Massachusetts General Hospital (MGH) miniature swine were used as a large animal model with defined MHC, and musculoskeletal grafts from the donor hind limb were transplanted heterotopically to the recipient femoral vessels. Allografts from MHC-mismatched donors treated with cyclosporine (n = 4) were rejected in less than 6 weeks by gross inspection and histologic sections. Allografts from MHC-matched, minor antigen mismatched donors not treated with cyclosporine (n = 4) were rejected between 9 and 12 weeks. Allografts from similarly matched donors treated with 12 days of cyclosporine (n = 7) showed no evidence of rejection until sacrifice between 25 and 47 weeks. Thus allograft tolerance was achieved between MHC-matched swine using a limited course of cyclosporine. Demonstration of limb tissue allograft survival in a large animal model without long-term immunosuppression represents an important step toward clinical transplantation.

Injection of allogeneic bone marrow cells into the portal vein of swine in utero.

The ability to safely manipulate the immune system of the developing fetus carries the hope of effective treatment strategies for certain congenital disorders that can be diagnosed during gestation. One possible intervention is the induction of specific transplantation tolerance to an adult donor who could provide tissue after birth without the need for immunosuppression. Although the introduction of allogeneic stem cells to a developing immune system has been shown to result in hematopoietic chimerism, donor-specific transplantation tolerance has not been demonstrated in a large animal model. In previous reports of in utero stem-cell transplantation, the cells were injected into the fetus by an intraperitoneal route. We sought to improve upon this technique of cell transplantation by developing a method for the safe delivery of allogeneic stem cells directly into the hepatic circulation of fetal swine. In the second phase of our study, we determined if adult allogeneic bone marrow cells delivered to the fetus by this intravascular route could result in result in hematopoietic chimerism and donor-specific transplantation tolerance. A method of successful intravascular injection was designed in which a laparotomy was performed on a sow at midgestation (50-55 days) to administer 1 cc of inoculum into the portal vein of each fetus using transuterine ultrasound guidance and a 25-gauge spinal needle. In one sow, 10 piglets were injected with saline to test safety, and 8 piglets were born. For transplantation of stem cells to the fetuses, donor bone marrow was harvested from a genetically defined miniature swine. In one sow the marrow was injected without T-cell depletion resulting in abortion. In the third sow, the marrow was depleted of T-cells to less than 0.01% using magnetic beads conjugated to anti-CD3 monoclonal antibodies. No chimerism was detected in these offspring. Only in the fourth sow where the T-cell depletion was reduced to about 1% of the cells in the inoculum did one animal demonstrate chimerism. This piglet showed reproducible blood chimerism (0.95% donor cells) detected by flow cytometry measurement of monoclonal antibodies to the donor MHC. In addition, this animal demonstrated hyporesponsiveness to donor lymphocytes in an MLR assay while reacting strongly to third-party stimulator cells. A split-thickness skin graft from the donor was accepted, and a third-party graft was rapidly rejected.

The tumescent technique: the effect of high tissue pressure and dilute epinephrine on absorption of lidocaine.

Injection of lidocaine into the subcutaneous tissues by the tumescent technique results in a delayed absorption of the local anesthetic and has allowed clinicians to exceed the maximum recommended dose of lidocaine without reported complications. However, little knowledge exists about the mechanisms that permit such high doses of lidocaine to be used safely with this technique. The presence of low concentration epinephrine and the increased tissue pressure resulting from the tumescent injection have both been implicated as important factors, but neither has been studied in patients whose results were not altered by the variability of the suction procedure. The purpose of this work was to determine the effect of tissue pressure during tumescent injection and presence of low concentration epinephrine on the absorption of lidocaine from subcutaneous tissues in human volunteers. Twenty healthy female human volunteers were randomized into four study groups. After body fat measurements, all subjects received an injection of 7 mg/kg of lidocaine into the subcutaneous tissues of both lateral thighs. The injected solution consisted of 0.1% lidocaine and 12.5 meq/liter sodium bicarbonate in normal saline with or without 1:1,000,000 epinephrine. Tissue pressure was recorded during injection using a specially designed double-barreled needle. The time required for injection was also recorded. Subjects in group 1 received lidocaine with epinephrine injected by a high-pressure technique. Group 2 subjects received lidocaine with epinephrine injected by a low-pressure technique. Group 3 subjects received lidocaine without epinephrine injected under high pressure. Group 4 subjects received lidocaine without epinephrine injected under low pressure. Following injection, sequential blood samples were drawn over a 14-hour period, and plasma lidocaine concentrations were determined by gas chromatography. No suction lipectomy was performed. Maximum tissue pressure during injection was 339 +/- 63 mmHg and 27 +/- 9 mmHg using high- and low-pressure techniques, respectively. Addition of 1:1,000,000 epinephrine, regardless of the pressure of injected fluid, significantly delayed the time to peak plasma concentration by over 7 hours. There was no significant difference in the peak plasma concentration of lidocaine among the four groups. Peak plasma concentrations greater than 1 mcg/ml were seen in 11 subjects. Epinephrine (1:1,000,000) significantly delays the absorption of lidocaine administered by the tumescent technique. High pressure generated in the subcutaneous tissues during injection of the solution does not affect lidocaine absorption. The delay in absorption may allow time for some lidocaine to be removed from the tissues by suction lipectomy. In addition, the slow rise to peak lidocaine concentration in the epinephrine groups may allow the development of systemic tolerance to high lidocaine plasma levels.

Use of swine model in transplantation of vascularized skeletal tissue allografts.

Permanent tolerance to vascularized skeletal tissue allografts can be induced in miniature swine with minor antigen differences using a 12-day course of CsA. Demonstration of skeletal tissue allograft survival in a large animal model without long-term immunosuppression represents an important step toward transplantation of skeletal tissue allografts in humans.

Complications and toxicities of implantable biomaterials used in facial reconstructive and aesthetic surgery: a comprehensive review of the literature.

The use of implantable biomaterials has become an integral part of aesthetic and reconstructive surgery of the face. Metals are used for fracture fixation devices, whereas polymers are used primarily for bone or soft-tissue substitution. This review of the scientific literature examines the risks and complications of these materials. First, we present an overview of commonly used materials. Second, we address general considerations of toxicity relevant to all biomaterials. Third, we present data from a large number of clinical series on the incidence of complications for individual materials used in specific applications.

Symposium "Implantable Materials in Facial Aesthetic and Reconstructive Surgery: Biocompatibility and Clinical Applications". American Society of Maxillofacial Surgeons. Montreal, Quebec, October 6, 1995.

On October 6, 1995, the American Society of Maxillofacial Surgeons sponsored a 1-day symposium entitled "Implantable Materials in Facial Aesthetic and Reconstructive Surgery: Biocompatibility and Clinical Applications." The symposium examined issues relating to the biocompatibility and clinical role of alloplastic materials commonly used for facial bone and soft-tissue replacement and augmentation. It provided a forum for the interaction of basic scientists, clinicians, and manufacturers. Clinical and laboratory data concerning a variety of implantable materials were presented and discussed. The program consisted of three parts. The first session was designed to provide historical and scientific background as well as perspective on legal issues surrounding the use of implantable biomaterials. The second session involved the presentation of clinical data on bone and bone substitutes for augmentation of the facial skeleton. The third session was devoted to clinical reports of bone and bone substitutes used for the reconstruction of cranial vault and cranial base skull defects.

Variables affecting the accuracy of stereotactic localization using computerized tomography.

Stereotactic localization using computerized tomography (CT) is increasingly employed to guide neurosurgical procedures in crucial areas of the brain such as the brain stem. This technique allows the surgeon to resect a lesion in its entirety while sparing critical areas of the brain. Thus, the parameters used for scanning should be selected for maximum accuracy. While the small pixel size of CT scanners suggests a high degree of precision in localization, there have been few systematic studies of this accuracy. The authors have studied the amount of error in localization created by variables such as CT scan thickness, interscan spacing, size of lesion, and method of computation when using the Brown-Roberts-Wells (BRW) stereotactic system. Over 1000 CT scans were made of a phantom composed of spheres of differing diameter and location. The CT slice thickness was varied from 1.5 to 5.0 mm, and interscan spacing was varied from 0.5 to 3.0 mm. The coordinates of the center of the spheres were calculated independently using the laptop computer supplied with the unit and also by a stereotactic computer which automatically calculates the center of the fiducials. The actual BRW coordinates of the sphere center were obtained using the phantom base and were then compared to the computer-calculated coordinates to determine error in localization. Variables with a significant effect on error included the scan thickness, interscan spacing, and sphere size. The mean error decreased 23% as the scan thickness decreased from 5.0 to 1.5 mm and 45% as the interscan spacing decreased from 3.0 to 0.5 mm. Mean error was greatest for the smallest sphere sizes. The two computational methods did not differ in error. This study suggests that, for critical areas of the brain or for small lesions, a scan thickness of 1.5 mm and interscan spacing of 0.5 mm should be employed.