Dennis von Heimburg

Human preadipocytes seeded on freeze-dried collagen scaffolds investigated in vitro and in vivo

Currently, there is no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, after tumor resection and in hereditary and congenital defects (e.g. Rombergs disease, Poland syndrome). The autologous transplantation of mature adipose tissue has poor results. In this study human preadipocytes of young adults were isolated and cultured. 10(6) preadipocytes were seeded onto collagen sponges with uniform 40 microm pore size and regular lamellar structure and implanted into immunodeficient mice. Collagen sponges without preadipocytes were used in the controls. Macroscopical impression, weight, thickness, histology, immunohistochemistry (scaffold structure, cellularity, penetration depth of the seeded cells) and ultrastructure were assessed after 24 h in vitro and after explantation at 3 and 8 weeks. Preadipocytes penetrated the scaffolds 24 h after seeding at a depth of 299+/-55 microm before implantation. Macroscopically after 3 and 8 weeks in vivo layers of adipose tissue accompanied by new vessels were found on all preadipocyte/collagen grafts. The control grafts appeared unchanged without vessel ingrowth. There was a significant weight loss of all grafts between 24 h in vitro and 3 weeks in vivo (p < 0.05), whereas there was only a slight weight reduction from week 3 to 8. The thickness decreased in the first 3 weeks (p < 0.05) in all grafts. The preadipocyte/collagen grafts were thinner but had a higher weight than the controls at this point in time. The histology showed adipose tissue and a rich vascularisation adherent to the scaffolds under a capsule. The control sponges contained only few cells and a capsule but no adipose tissue. Human-vimentin positive cells were found in all preadipocyte/collagen grafts but not in the controls, penetrating 1188+/-498 microm (3 weeks) and 1433+/-685 microm (8 weeks). Ultrastructural analysis showed complete in vivo differentiation of viable adipocytes in the sponge seeded with preadipocytes. Formation of extracellular matrix was more pronounced in the preadipocyte/collagen grafts. The transplantation of isolated and cultured preadipocytes within a standardised collagen matrix resulted in well-vascularised adipose-like tissue. It is assumed that a pore size greater than 40 microm is required, as preadipocytes enlarge during differentiation due to incorporation of lipids.

Influence of different biodegradable carriers on the in vivo behavior of human adipose precursor cells.

The correction of soft-tissue defects presents a challenge in plastic and reconstructive surgery. The implantation of isolated and culture-expanded adipose precursor cells is a solution to this problem because these cells differentiate into adipocytes when implanted in vivo. Appropriate scaffolds are needed in soft-tissue engineering to allow the differentiation of precursor cells. The optimal carrier needs to be defined. In this study, human preadipocytes were isolated and cultured. Three different carrier materials were seeded with 106 preadipocytes each and implanted in 42 nude mice. Sponges and nonwoven carriers based on hyaluronic acid modified by esterification (HYAFF 11) were compared with collagen sponges. Scaffolds without cells served as negative controls in the same animal. After 3 and 8 weeks, the grafts were explanted. Macroscopic appearance, weight, thickness, microscopy, immunohistochemistry, and TEM (scaffold structure, cellularity, penetration depth of the seeded cells, vascularization) were assessed and evaluated for differences in scaffold-cell interactions.Preadipocytes differentiated earlier in vitro when attached to HYAFF 11 scaffolds than to other carrier materials. Macroscopically, all preadipocyte constructs were yellowish and well vascularized, and the controls were white and avascular. Vessel formation was more pronounced around mature adipocytes. Microscopically, HYAFF 11 constructs showed a higher cell density than collagen constructs. The pores of the sponges contained more differentiated adipocytes than the nonwoven carriers, whereas the undifferentiated preadipocytes were more numerous in the nonwoven material. Penetration of adipose precursor cells was deeper and more homogeneous in HYAFF 11 scaffolds. Electron microscopy demonstrated well-differentiated adipocytes and large amounts of extracellular matrix in HYAFF 11 sponges.HYAFF 11 sponges supported the expansion and differentiation of the adipose precursor cells. This carrier is superior to the nonwoven carrier with regard to adipocyte differentiation and superior to the collagen sponge with regard to cellularity. This is a promising method for the reconstruction of soft-tissue defects. Modifications of the scaffold (larger pore size and coating with adipogenic factors) will be examined in further experiments.

Comparison of Viable Cell Yield from Excised versus Aspirated Adipose Tissue

The correction of soft-tissue defects by adipose tissue transplantation often produces poor and unpredictable results. The implantation of isolated and cultured preadipocytes offers a solution to this problem since these cells differentiate into adipocytes when implanted in vivo. A field of major interest is to maximize the yield of preadipocytes isolated from adipose tissue showing only low contamination with other cell types. Aspiration and excision are two concurrent clinical ways of harvesting adipose tissue for the isolation of preadipocytes. This tissue is usually discarded after surgery. In this study, the yield of preadipocytes obtained from liposuction material was compared to that of excised adipose tissue. Furthermore, we determined the loss of precursor cells if isolation of preadipocytes was delayed for 24 h. Preadipocytes were isolated from the stromal cell fraction of human subcutaneous adipose tissue samples. Harvesting of adipose tissue by suction was performed according to the Coleman procedure (manually applied negative pressure using a 10-ml syringe with a blunt tip cannula). Isolation was either carried out within 60 min after extraction or after storage for 24 h in culture medium at 4°C. Isolated preadipocytes were cultured for 24 h, trypsinized and counted in a Neubauer chamber. Our results show clearly that the yield of preadipocytes isolated from liposuction material (within 60 min after extraction and after 24 h of storage) is higher than the cell yield from excised adipose tissue. Overnight storage for 24 h leads to a significant loss of preadipocytes in excised tissue but not in liposuction material. The high yield of cells isolated from liposuction material proves that extraction by suction does not damage the stromal cell fraction in the adipose tissue. If cell isolation is not performed immediately after the operation, liposuction material is clearly the better alternative for storage.

Oxygen consumption in undifferentiated versus differentiated adipogenic mesenchymal precursor cells

To date, no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, tumor resections or in congenital defects is available. A biohybrid composed of viable adipose precursor cells and an optimised matrix could help towards a solution. Morphologically, preadipocytes resemble fibroblasts and have not yet built a large cytoplasmic lipid droplet as found in differentiated adipocytes. Additionally, preadipocytes are smaller than mature adipocytes allowing a quicker revascularization after transplantation. Furthermore, transplanted preadipocytes can form adipose tissue in vivo whereas the transplantation of mature adipocytes often gives poor results, i.e. oil cysts or shrinkage of the transplant. Since these observations point to differences in metabolic activity between preadipocytes and adipocytes, we investigated the oxygen consumption of preadipocytes stimulated to undergo differentiation, and fibroblasts, by measuring the respiration with a Clark-type oxygen electrode. Preadipocytes had a significantly lower oxygen consumption than mature adipocytes. This advantage in respiration and the better revascularization of undifferentiated adipose tissue cells allow the development of innovative transplants and point to preadipocytes as promising tool to improve transplantations in adipose tissue reconstruction.

Autologous In Vivo Adipose Tissue Engineering in Hyaluronan-Based Gels—A Pilot Study

BACKGROUND There is a major clinical need for strategies for adequately reconstructing the soft tissue defects found after deep burns, tumor resection, or trauma. A promising solution is adipose tissue engineering with preadipocytes, stem-cell derived precursors of the adipose tissue, implanted within biomaterials. This pilot study evaluated hyaluronan gels mixed with autologous undifferentiated preadipocytes in a pig model for their potency to generate new fat. MATERIALS AND METHODS Preadipocytes were isolated from intra-abdominal pig fat by collagenase digestion, plated on fibronectin-coated culture dishes in Dulbeccos modified Eagle medium/Hams F12 (Biochrom, Berlin, Germany) combined with 10% pig serum, expanded, and mixed with hyaluronan gel. Two types of gels with varying degrees of amidation of the carboxyl groups were tested (HYADD3, HYADD4). Cell-loaded gels and unseeded controls were injected subcutaneously into the ears of three pigs, explanted at 6 wk, and analyzed histologically. RESULTS Both cell-loaded specimens were detected macroscopically. They demonstrated a slight volume effect with limited stability after 6 wk. Unloaded HYADD3 and HYADD4 controls could not be identified at the time of explantation. Histology of HYADD3 revealed islets of mature adipocytes and vessels embedded in fat tissue surrounded by gel. In contrast, no fat formation was found in HYADD4 gels when implanted in the ear. CONCLUSIONS Histological findings demonstrate that HYADD3 is a promising gel for generating adipose tissue. Even though HYADD3 might be a potential material for the reconstruction of small tissue defects, the question remains as to whether the adipose tissue within the gel is attributable to preadipocyte maturation or ingrowth from neighboring tissue.

TIMP-1, MMP-2, MMP-9, and PIIINP as serum markers for skin fibrosis in patients following severe burn trauma.

&NA; The wound‐healing process of patients with severe burns often leads to the formation of extensive fibrotic scars. In this study, serum concentrations of tissue inhibitors of metalloproteinase‐1 (TIMP‐1), matrix metalloproteinase‐2 (MMP‐2), matrix metalloproteinase‐9 (MMP‐9), and amino‐terminal propeptide of procollagen type III (PIIINP) were measured by enzyme‐linked immunosorbent assay as markers for excessive cicatrization in 22 patients with acute burn injuries. All patients were followed up for 6 months to determine a fibrotic reaction during the wound‐healing process after operative treatment using the Burn Scar Index. Blood samples were drawn immediately before the operation; at postoperative days 1, 3, 7, and 14; and 1, 3, and 6 months after the operation. Twenty patients who underwent elective plastic surgical operations served as the control group. There was a significant increase (p < 0.05) of TIMP‐1 in the burned patients by the third postoperative day. Later in the follow‐up period, the serum concentrations remained at a significantly elevated level (p < 0.05) compared with preoperative values. In comparison with the control group, the postoperative serum concentrations of TIMP‐1 of the burned patients were significantly higher (p < 0.05) at any time and correlated with the total body surface area burned at the third and seventh postoperative days (p < 0.05; &ggr;2 = 0.46 versus r2 = 0.53) and the Burn Scar Index after 6 months (p < 0.05; r2 = 0.65). Serum levels of MMP‐2 and MMP‐9 showed a significant elevation (p < 0.05) only between postoperative days 3 and 14 in patients with burn wounds. PIIINP increased significantly (p < 0.05) in the sera of the burned patients at postoperative day 3 and remained significantly elevated up to 6 months after injury. At any time after trauma, PIIINP serum levels were significantly higher (p < 0.05) in the burned patients than in the control group and correlated with the total body surface area burned at postoperative days 3 and 7 (p< 0.05; r2 = 0.41 versus r2 = 0.44) and the Burn Scar Index after 6 months (p < 0.05; r2 = 0.5). Obviously, the physiological balance between matrix metalloproteinases and their endogenous inhibitors is disturbed after burn trauma. The elevated systemic TIMP‐1 concentration might contribute to tissue fibrosis, leading to pathological scar formation. The increase of PIIINP after thermal trauma indicates a fibrogenic component of wound healing. (Plast. Reconstr. Surg. 111: 1423, 2003.)

Biomaterials for adipose tissue engineering

There is high clinical need for an adequate reconstruction of soft tissue defects as found after tumor resections, deep burns or severe trauma. A promising solution for these defects is adipose tissue engineering, with adult stem cells of the adipose tissue, implanted on 3D biomaterials. These adipogenic precursor cells survive ischemia better than mature adipocytes and have the potency to proliferate and differentiate into fat cells after transplantation. They can be yielded from excised adipose tissue or liposuction material. When preadipocytes are seeded on carriers for the generation of adipose tissue, chemical composition, mechanical stability and 3D architecture of the construct are crucial factors. They ensure cellular penetration into the construct, sufficient proliferation on the material and full differentiation inside the construct after transplantation. In hydrogels, it is especially the use and combination of growth factors that determine the overall outcome of the applied biopolymer. Over recent years, in vivo trials in particular have allowed significant insights into the potential, the perspectives, but also the current difficulties and draw-backs in adipose tissue engineering. This review focuses on the main strategies in adipose tissue regeneration, compares the various materials that have been used as carrier matrices so far and considers them in light of the challenges they have yet to meet.

Hyperbaric oxygen treatment in deep frostbite of both hands in a boy

UNLABELLED An 11-year-old boy in good general health conditions suffered deep frostbite on six fingers while he was working without gloves as a beater during a hunt in Poland at an outdoor temperature of -32 degrees C over a 4 h-period. Three days later he was first seen by a physician who planned to amputate the affected fingers. The patient was transferred by his family to our University Hospital in Aachen, Germany. We found third degree frostbite on four fingers of the right and on two fingers of the left hand. Because of the late beginning of the therapy, the patient was treated by HBO(2) according to the Marx-schema for problem wounds (2,4 bar, total time at depth: 90 min, alternations of 100% O(2) and air breathing). HBO(2)-treatment was repeated daily for 14 days. No adverse events were recorded during the course of therapy. A total recovery of the severe frostbite was observed after 14 days of HBO(2)-treatment. Twenty-eight months after the injury the patient reports fully regained sensibility and no pain. The plain X-ray after this period showed no premature closure of the epiphyses or sclerosis of the metaphyses. CONCLUSIONS Because of the low risk associated with HBO(2), and its potential therapeutic efficiency, HBO(2) should be recommended as adjunct therapy in the treatment of deep frostbite.

Applicability of the dyes CFSE, CM-DiI and PKH26 for tracking of human preadipocytes to evaluate adipose tissue engineering.

Adipose tissue engineering with preadipocyte-loaded scaffolds requires adequate tracking of preadipocytes to allow evaluation and quantification of cell proliferation, expansion and differentiation in three-dimensional systems. To differentiate between graft and host cells, labeling of preadipocytes before implantation and tracking of these cells until harvest would be useful. Immunohistochemistry enables the differentiation between cells of different species but is time-consuming, expensive, elaborate, and not applicable for autologous transplantation. So far, there is no published method to use externally applied dyes for tracking of human preadipocytes in adipose tissue engineering. We tested the cell dyes PKH26, CM-DiI, and CFSE to analyze their applicability for labeling human preadipocytes. CM-DiI had toxicity levels of 45–70%, while 3–4% proliferating cells were stained on day 35. CFSE revealed clear cytoplasmic coloring in proliferating cells with 5–6% stained cells after 35 days and toxicity ranging from 55 to 90% dead cells. PKH26 demonstrates lowest levels of toxicity and best labeling results after 4 weeks in proliferating preadipocytes in monolayer. Although none of the dyes showed long-lasting labeling during proliferation, all three dyes demonstrated permanent staining in differentiated cells. The results reveal the problems of preadipocyte tracking with fluorescent dyes but justify the dye application for limited time periods.

Pathogenic role of interleukin-6 in the development of sepsis. Part Ii: Significance of anti-interleukin-6 and anti-soluble interleukin-6 receptor-α antibodies in a standardized murine contact burn model

ObjectiveThe in vivo effects of anti-interleukin-6 (anti-IL-6) and anti-interleukin-6-&agr; receptor (anti-IL-6R) monoclonal antibodies on immune response and survival rate of a burn with subsequent infection were assessed. SubjectsTen-week-old C 57 BL/6J mice received a standardized contact burn; 48 hrs later endotoxin (LPS) was injected intraperitoneally to induce systemic inflammation. Ten different groups were studied. Groups I–IV sustained a burn and/or a LPS-stimulus but did not receive any anti-cytokines and served as controls. Treatment groups V–X sustained the same injuries but also received anti-IL-6 and anti-IL-6R intravenously either before or after the LPS stimulus. In a further part of the study, a lethal dose of LPS was injected (LPS-LD100 group) followed by an injection of anti-IL-6 antibody and/or anti-IL-6R antibody. MeasurementsSerum concentrations of IL-6, tumor necrosis factor (TNF)-&agr;, interferon (IFN)-&ggr;, and white blood cell and platelet counts were determined, and the survival rate over a 2-wk period was assessed. ResultsTreatment with anti-IL-6 slightly decreased the inflammatory response when it was given before or after LPS application. The inflammatory response was not decreased after treatment with anti-IL-6R. In the groups that received a combination of anti-IL-6 and anti-IL-6R, there was a significant reduction of the inflammatory response. This was more pronounced when the anti-cytokines were applied after LPS application. A significant reduction in mortality could be shown with both antibodies in the treatment groups and the groups that had received a lethal dose of LPS (LPS-LD100 group). ConclusionsIL-6 has a low inflammatory potential, and IL-6R has no inflammatory potential by itself. In contrast, the IL-6/IL-6R complexes have a higher inflammatory potential. Mortality could be reduced by each antibody alone as well as by the combination, supporting the hypothesis that the inflammatory and lethal potentials of IL-6 are not identical. The study suggests that the use of antibodies against IL-6 or IL-6R is effective in the prevention of systemic inflammation in a murine burn model.