Johannes Frank

Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro

INTRODUCTION Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. MATERIALS AND METHODS This in vitro study investigates cell seeding efficiency, metabolism, gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX), synthetic alpha-TCP (Biobase), synthetic beta-TCP (Vitoss), synthetic beta-TCP (Chronos), processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds, respectively. On days 2, 6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. RESULTS The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; p<0.05) followed by Chronos (47.5+/-19.5, p<0.05), Actifuse ABX (19.1+/-4.4), Biobase (15.7+/-9.9), Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters, depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds, beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. DISCUSSION Our results indicate that various bone-graft substitutes influence cell seeding efficiency, metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro, which may be important for bony integration in the clinical setting.

Composite tissue allotransplantation of the hand and face: a new frontier in transplant and reconstructive surgery

Each year an estimated 7‐million people in the USA need composite tissue reconstruction because of surgical excision of tumors, accidents and congenital malformations. Limb amputees alone comprise over 1.2 million of these. This figure is more than double the number of solid organs needed for transplantation. Composite tissue allotransplantation in the form of hand and facial tissue transplantation are now a clinical reality. The discovery, in the late 1990s, that the same immunotherapy used routinely in kidney transplantation was also effective in preventing skin rejection made this possible. While these new treatments seem like major advancements most of the surgical, immunological and ethical methods used are not new at all and have been around and routinely used in clinical practice for some time. In this review of composite tissue allotransplantation, we: (i) outline the limitations of conventional reconstructive methods for treating severe facial disfigurement, (ii) review the history of composite tissue allotransplantation, (iii) discuss the chronological scientific advances that have made it possible, (iv) focus on the two unique clinical scenarios of hand and face transplantation, and (v) reflect on the critical issues that must be addressed as we move this new frontier toward becoming a treatment in mainstream medicine.

Investigation of risk acceptance in facial transplantation.

Background: The surgical techniques necessary to transplant a human face are well established, and the early success of human hand transplants suggests that the immunological hurdles of transplanting human facial tissues have largely been overcome. Therefore, it is the ethical barriers that pose the greatest challenge to performing facial transplantation. At the center of the ethical debate is the question, “Do the risks posed by the life-long immunosuppression that a recipient would have to take justify the benefits of receiving a face transplant?” In this study, the authors answer this question by assessing the degree of risk individuals would be willing to accept to receive a face transplant. Methods: To quantitatively assess risks versus benefits in facial transplantation, the authors developed the Louisville Instrument for Transplantation, or LIFT, which contains 237 standardized questions. Respondents in three study populations (healthy individuals, n = 150; organ transplant recipients, n = 42; and individuals with facial disfigurement, n = 34) were questioned about the extent to which they would trade off specific numbers of life-years, or sustain other costs, in exchange for receiving seven different transplant procedures. Results: The authors found that the three populations would accept differing degrees of risk for the seven transplant procedures. Organ transplant recipients were the most risk-tolerant group, while facially disfigured individuals were the least risk tolerant. All groups questioned would accept the highest degree of risk to receive a face transplant compared with the six other procedures. Conclusions: This study presents an empirical basis for assessing risk versus benefit in facial transplantation. In doing so, it provides a more solid foundation upon which to introduce this exciting new reconstructive modality into the clinical arena.

In vivo effect of hyperbaric oxygen on wound angiogenesis and epithelialization

Hyperbaric oxygen (HBO) therapy is increasingly being used in different areas of medical practice. While demonstrated to be effective in several settings, its mechanism of action is not well understood. In the present study, we determined the effects of HBO on wound epithelialization and neovascularization in an in vivo hairless mouse ear “impaired” wound model. To impair wound healing, macrophages were depleted by pretreatment with iota‐carrageenan. Wound epithelialization and neovascularization were measured using intravital microscopy and computerized planimetry. Metalloproteinase‐2 (MMP‐2), MMP‐9, tissue inhibitor of metalloproteinase‐1 (TIMP‐1), and tumor necrosis factor‐α (TNF‐α) were measured on days 2 and 7 using immunohistochemistry. In nonimpaired healing wounds, the rate of epithelialization and neovascularization was significantly accelerated in the groups treated with HBO. Time to wound closure was significantly delayed in impaired compared with nonimpaired healing wounds and HBO treatment completely reversed this delay. Neither HBO treatment nor macrophage depletion caused significant alterations in MMP‐2 expression in wounds. In contrast, TNF‐α, MMP‐9, and TIMP‐1 were significantly up‐regulated in the impaired healing group receiving HBO treatment. These results show that HBO therapy effectively reversed the negative effect exerted by macrophage reduction on wound epithelialization and neovascularization. This beneficial effect could be due to stimulation of TNF‐α production and, to a lesser degree due to release of metalloproteinases.

Establishment and characterization of the Masquelet induced membrane technique in a rat femur critical‐sized defect model

The Masquelet induced membrane technique for reconstructing large diaphyseal defects has been shown to be a promising clinical treatment, yet relatively little is known about the cellular, histological and biochemical make‐up of these membranes and how they produce this positive clinical outcome. We compared cellular make‐up, histological changes and growth factor expression in membranes induced around femur bone defects and in subcutaneous pockets at 2, 4 and 6 weeks after induction, and to the periosteum. We found that membranes formed around bone defects were similar to those formed in subcutaneous pockets; however, both were significantly different from periosteum with regard to structural characteristics, location of blood vessels and overall thickness. Membranes induced at the femur defect (at 2 weeks) and in periosteum contain mesenchymal stem cells (MSCs; STRO‐1+) which were not found in membranes induced subcutaneously. BMP‐2, TGFβ and VEGF were significantly elevated in membranes induced around femur defects in comparison to subcutaneously induced membranes, whereas SDF‐1 was not detectable in membranes induced at either site. We found that osteogenic and neovascular activity had mostly subsided by 6 weeks in membranes formed at both sites. It was conclude that cellular composition and growth factor content in induced membranes depends on the location where the membrane is induced and differs from periosteum. Osteogenic and neovascular activity in the membranes is maximal between 2 and 4 weeks and subsides after 6. Based on this, better and quicker bone healing might be achieved if the PMMA cement were replaced with a bone graft earlier in the Masquelet technique. Copyright

Psychosocial considerations in facial transplantation

The human face and facial transplantation have long captured the interest and imagination of scientists, the media and the lay public. The face is central to our identity, and our communication with the outside world. It is this great importance we attach to our face that makes facial disfigurement such a devastating condition. Facial transplantation could provide an excellent alternative to current treatments for facial disfigurement caused by burns, trauma, cancer extirpation or congenital birth defects. Herein we discuss some of the principal psychosocial considerations which have preceded the clinical introduction of facial transplantation, and which continue today after cases have been performed world-wide.

In Vivo Effect of Tumor Necrosis Factor Alpha on Wound Angiogenesis and Epithelialization

AbstractBackground:The role of tumor necrosis factor alpha (TNF-α) in wound healing is unclear and the results are contradictory. In vivo, TNF-α induces vessel growth, an important step in promoting wound healing. However, a reduced amount of collagen, hydroxyproline, and granulation tissue was found after TNF-α treatment. It is also unknown if this is a direct effect, by influencing cells involved in wound healing, or an indirect effect due to a negative or positive effect on cells such as macrophages.Material and Methods:The current study was undertaken to test the effect of TNF-α on wound epithelialization and neovascularization in vivo. In the first experiment, standardized full-thickness dermal wounds (2.25 mm diameter, 0.125 mm depth) were created on the dorsum of the ears of male hairless mice. The wounds were treated either with TNF-α (100 ng/ml, 1 µg/ml, 5 µg/ml; n = 10 per group), monoclonal TNF-α antibody (10 µg/ml; n = 10), or vehicle (n = 10). Wound epithelialization and neovascularization were analyzed every 3rd day by intravital microscopy until complete healing.In a second experiment, the same wound model was used, but in order to impair the healing process, macrophages were depleted. To reduce macrophages, two out of four groups (n = 10 per group) were pretreated with iota-carrageenan (MR groups), and the other two groups received only saline (N groups). One N group and one MR group were treated with TNF-α (1 µg/ml). The other N group and MR group received vehicle only (carboxymethylcellulose). Using intravital microscopy and computerized planimetry, wound epithelialization and neovascularization were measured every 3rd day until complete healing. Immunohistochemistry was performed to detect TNF-α, macrophages, fibronectin, and vitronectin receptors.Results:In the first experiment the wounds treated with 1 µg/ml healed significantly earlier than controls (13.9 ± 2 vs. 17.3 ± 2.8 days, respectively; p < 0.05). Epithelialization in the antibody group was significantly slower compared to controls (20.1 ± 2 days; p < 0.05). Neovascularization was significantly enhanced in the group treated with 1 µg/ml TNF-α (p < 0.05). In the second experiment the wounds treated with TNF-α were significantly earlier epithelialized (13.1 ± 0.6) and vascularized (16.0 ± 0.5) compared to controls (16.8 ± 0.4 vs. 17.6 ± 0.5; p < 0.05). Wound closure was significantly delayed in the MR group treated with vehicle only (20.4 ± 1) and equal to controls in the MR group treated with TNF-α (16.8 ± 0.6).Conclusion:The results demonstrate the TNF-α accelerates wound epithelialization and neovascularization in this in vivo model. TNF-α is able to compensate for the negative effect of macrophage reduction and seems to have a direct effect on the wound-healing process.

Risk Acceptance in Composite Tissue Allotranplantation Reconstructive Procedures

AbstractComposite tissue allotransplantation (CTA) has recently emerged as a new therapeutic modality to reconstruct major tissue defects of the face, larynx, and extremities. However, because the risk-versus-benefit ratio for CTA procedures has not yet been defined, the decision as to whether or not to perform these new reconstructive techniques is based on subjective opinions rather than objective data. To address this problem, the authors developed a reliable and valid instrument to objectively assess the relative risk that individuals are willing to accept in order to receive the benefits of various CTA procedures.

The hairless mouse ear: an in vivo model for studying wound neovascularization

Microvascular ingrowth into damaged tissue is an essential component of the normal healing process. In fact, wound therapy is often aimed at promoting neovascularization. However, little is known about the mechanisms that regulate microvascular ingrowth into a healing wound. This limited knowledge is largely due to the lack of adequate models in which microvascular ingrowth can be quantitatively analyzed throughout the healing process. To address this deficiency, we developed a model in which a wound was created on the ear of the hairless mouse—a well established model for directly viewing and measuring skin microcirculation. While the animals were under ketamine and xylazine anesthesia, 2.25 mm diameter full‐thickness wounds were created on the dorsum of hairless mouse ears down to but not including the cartilage (0.125 mm depth). With the use of video microscopy and computer‐assisted digitized planimetry, the precise epithelial and neovascular wound edge was viewed and measured regularly throughout healing. Therefore, this model can provide objective data on wound epithelialization and neovascularization throughout healing. This model was used to examine the effect of topical wound agents on epithelialization and neovascularization. Differential effects by these anti‐microbial agents on these two processes were observed, which suggests clinical implications for their use.

Composite Tissue Allotransplantation (CTA): Current Status and Future Insights

Approximately 7 million individuals (over 1 million amputees) require complex reconstructive procedures in the United States each year. The recent success of clinical composite tissue allotransplantation, attests to the fact that composite tissue allografts have tremendous potential in these life-enhancing reconstructions.This review summarizes the initial outcomes of the first four human hand transplants, together with those of the first larynx, bone, knee, nerve and tendon transplants, with special emphasis on the operative technique, graft survival and functional outcomes. The May 2000 Louisville symposium, where these results were presented was undoubtedly a milestone in the history of modern composite tissue allotransplantation. It set the stage for reconstructive and transplant surgeons, researchers, physiotherapists, patients and patient advocates and members of the community to convene and discuss major advances in current composite tissue allotransplantation. The symposium underscored the vital importance of objective evaluation of the status of composite tissue allotransplantation by frank dissemination of details of clinical results and complications of the transplants performed thus far.The composite tissue allotransplantation area is among the newest of transplant areas. The immunology of composite tissue allografts is complex, making tolerance more difficult to achieve than organ tolerance. It needs to be emphasized that any episodes of acute rejection should be prevented for perfect restoration of function and to minimize the risk of chronic rejection in composite tissue allografts. Efficacious, safe and ethical clinical tolerance protocols could improve patient acceptance of composite tissue allografts by providing an alternative to chronic immunosuppression.