Peter M. Vogt

Skin tissue generation by laser cell printing

For the aim of ex vivo engineering of functional tissue substitutes, Laser‐assisted BioPrinting (LaBP) is under investigation for the arrangement of living cells in predefined patterns. So far three‐dimensional (3D) arrangements of single or two‐dimensional (2D) patterning of different cell types have been presented. It has been shown that cells are not harmed by the printing procedure. We now demonstrate for the first time the 3D arrangement of vital cells by LaBP as multicellular grafts analogous to native archetype and the formation of tissue by these cells. For this purpose, fibroblasts and keratinocytes embedded in collagen were printed in 3D as a simple example for skin tissue. To study cell functions and tissue formation process in 3D, different characteristics, such as cell localisation and proliferation were investigated. We further analysed the formation of adhering and gap junctions, which are fundamental for tissue morphogenesis and cohesion. In this study, it was demonstrated that LaBP is an outstanding tool for the generation of multicellular 3D constructs mimicking tissue functions. These findings are promising for the realisation of 3D in vitro models and tissue substitutes for many applications in tissue engineering. Biotechnol. Bioeng. 2012; 109:1855–1863.

Tissue Engineered Skin Substitutes Created by Laser-Assisted Bioprinting Form Skin-Like Structures in the Dorsal Skin Fold Chamber in Mice

Tissue engineering plays an important role in the production of skin equivalents for the therapy of chronic and especially burn wounds. Actually, there exists no (cellularized) skin equivalent which might be able to satisfactorily mimic native skin. Here, we utilized a laser-assisted bioprinting (LaBP) technique to create a fully cellularized skin substitute. The unique feature of LaBP is the possibility to position different cell types in an exact three-dimensional (3D) spatial pattern. For the creation of the skin substitutes, we positioned fibroblasts and keratinocytes on top of a stabilizing matrix (Matriderm®). These skin constructs were subsequently tested in vivo, employing the dorsal skin fold chamber in nude mice. The transplants were placed into full-thickness skin wounds and were fully connected to the surrounding tissue when explanted after 11 days. The printed keratinocytes formed a multi-layered epidermis with beginning differentiation and stratum corneum. Proliferation of the keratinocytes was mainly detected in the suprabasal layers. In vitro controls, which were cultivated at the air-liquid-interface, also exhibited proliferative cells, but they were rather located in the whole epidermis. E-cadherin as a hint for adherens junctions and therefore tissue formation could be found in the epidermis in vivo as well as in vitro. In both conditions, the printed fibroblasts partly stayed on top of the underlying Matriderm® where they produced collagen, while part of them migrated into the Matriderm®. In the mice, some blood vessels could be found to grow from the wound bed and the wound edges in direction of the printed cells. In conclusion, we could show the successful 3D printing of a cell construct via LaBP and the subsequent tissue formation in vivo. These findings represent the prerequisite for the creation of a complex tissue like skin, consisting of different cell types in an intricate 3D pattern.

Preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement and publication bias

Publication bias is a major problem in evidence based medicine. As well as positive outcome studies being preferentially published or followed by full text publication authors are also more likely to publish positive results in English-language journals. This unequal distribution of trials leads to a selection bias in evidence l level studies, like systematic reviews, meta-analysis or health technology assessments followed by a systematic failure of interpretation and in clinical decisions. Publication bias in a systematic review occurs mostly during the selection process and a transparent selection process is necessary to avoid such bias. For systematic reviews/meta-analysis the PRISMA-statement (formerly known as QUOROM) is recommended, as it gives the reader for a better understanding of the selection process. In the future the use of trial registration for minimizing publication bias, mechanisms to allow easier access to the scientific literature and improvement in the peer review process are recommended to overcome publication bias. The use of checklists like PRISMA is likely to improve the reporting quality of a systematic review and provides substantial transparency in the selection process of papers in a systematic review.

Acute and Overuse Injuries Correlated to Hours of Training in Master Running Athletes

Background: The goal of the study was to determine the rate of running-associated tendinopathy in light of the amount of time training and other risk factors. Materials and Methods: 291 elite runners (average age 42 ± 9 years) who ran an average of 65.2 ± 28.3 km/week were included with an overall distance of 9,980,852 km (34,416 km/athlete). Descriptive statistics with Chi 2 -Test, Fisher-Exact-Test and Mann-Whitney-Test were used to calculate relative risks (RR). Results: The overall injury rate was 0.08/1000 km (2.93/athlete). Overuse injuries (0.07/1000 km) were more frequent than acute injuries (0.01/1000 km). Achilles tendinopathy was the predominant injury (0.02/1000 km) followed by anterior knee pain (0.01/1000 km), and shin splints (0.01/1000 km). Achilles tendon rupture was rarely encountered (0.001/1000 km). At some time, 56.6% of the athletes had an Achilles tendon overuse injury, 46.4% anterior knee pain, 35.7% shin splints, and 12.7% had plantar fasciitis. Mid-portion Achilles tendinopathy was more common (0.01/1000 km) than insertional (0.005/1000 km). An asphalt running surface decreased mid-portion tendinopathy risk (RR 0.47, p = 0.02). In contrast, sand increased the relative risk for mid-portion Achilles tendinopathy tenfold (RR 10, CI 1.12 to 92.8, p = 0.01). Runners with more than 10 years experience had an increased risk (RR 1.6, p = 0.04) for Achilles tendinopathy. Conclusion: Achilles tendinopathy is the most common running-associated tendinopathy followed by runners knee and shin splints.

Percutaneous Collagen Induction Therapy: An Alternative Treatment for Scars, Wrinkles, and Skin Laxity

Background: Skin laxity, rhytides, and photoaging are generally treated by ablative procedures that injure or destroy the epidermis and its basement membrane, at least in the beginning, and subsequently lead to fibrosis of the papillary dermis. The ideal treatment would be to preserve the epidermis and promote normal collagen and elastin formation in the dermis. Percutaneous collagen induction takes us closer to this ideal. Methods: The authors performed a retrospective analysis of 480 patients in South Africa and Germany with fine wrinkles, lax skin, scarring, and stretch marks treated with percutaneous collagen induction using the Medical Roll-CIT to produce tighter, smoother skin. Most patients had only one treatment, but some have had as many as four treatments. Patients were prepared with topical vitamin A and C cosmetic creams for a minimum of 4 weeks preoperatively. Results: On average, patients in Germany rated their improvement between 60 and 80 percent better than before the treatment. Histologic examination was carried out in 20 patients and showed a considerable increase in collagen and elastin deposition at 6 months postoperatively. The epidermis demonstrated 40 percent thickening of stratum spinosum and normal rete ridges at 1 year postoperatively. Conclusions: Percutaneous collagen induction was started in 1997 and has proved to be a simple and fast method for safely treating wrinkles and scars. As opposed to ablative laser treatments, the epidermis remains intact and is not damaged. For this reason, the procedure can be repeated safely and is also suited to regions where laser treatments and deep peels cannot be performed.

Dry, Moist, and Wet Skin Wound Repair

Effects of wet (saline in a vinyl chamber), moist (hydrocolloid dressing), and dry (sterile gauze dressing) environments on wound repair were studied in a porcine partial-thickness wound model. Chambers were exchanged and refilled daily with normal saline containing penicillin G (100 U/ml) and streptomycin (100 µg/ml). Hydrocolloid and gauze dressings were kept in place until biopsy of the wound site. Wounds in wet, moist, and dry environments were completely epithelialized on days 6, 7, and 8, respectively. Thickness of the epidermis in wet, moist, and dry wounds was 204 ± 23, 141 ± 12, and 129 ± 18 (mean ± SEM), respectively. Moist wounds had more subepidermal inflammatory cells than wet wounds. In comparison to dry wounds, the moist or the wet healing environment resulted in less necrosis and faster and better quality of healing in the formation of the newly regenerated epidermis.

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP‐2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1–7 after wounding. The number of active MMP‐2 species detectable in wound fluid was greatest during days 4–6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP‐9 (92 kDa, Type V pro‐collagenase). In contrast to MMP‐2, the active form of this enzyme was never detected. The presence of MMP‐1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1–6 post‐injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds.

Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit.

Defects of peripheral nerves still represent a challenge for surgical nerve reconstruction. Recent studies concentrated on replacement by artificial nerve conduits from different synthetic or biological materials. In our study, we describe for the first time the use of spider silk fibres as a new material in nerve tissue engineering. Schwann cells (SC) were cultivated on spider silk fibres. Cells adhered quickly on the fibres compared to polydioxanone monofilaments (PDS). SC survival and proliferation was normal in Live/Dead assays. The silk fibres were ensheathed completely with cells. We developed composite nerve grafts of acellularized veins, spider silk fibres and SC diluted in matrigel. These artificial nerve grafts could be cultivated in vitro for one week. Histological analysis showed that the cells were vital and formed distinct columns along the silk fibres. In conclusion, our results show that artificial nerve grafts can be constructed successfully from spider silk, acellularized veins and SC mixed with matrigel.

Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration

Abstract.  Objective: In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20‐mm sciatic nerve defect in rats. Materials and methods: We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance. Results: Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S‐100 and neurofilament indicated existence of Schwann cells and axonal re‐growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell‐free constructs based on vein and spider silk was highly effective in nerve regeneration. Conclusion: We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re‐growth in a long‐distance model for peripheral nerve regeneration.

Transplantation of olfactory ensheathing cells enhances peripheral nerve regeneration after microsurgical nerve repair

While axonal regeneration is more successful in peripheral nerve than in the central nervous system, it is by no means complete and research to enhance peripheral nerve regeneration is clinically important. Olfactory ensheathing cells (OECs) are known to enhance axonal regeneration and to produce myelin after transplantation. In contrast to Schwann cells their migratory potential and ability to penetrate glial scars is higher. This study evaluated the effect of OEC transplantation on microsurgically repaired sciatic nerves. Rat sciatic nerves were transected followed by microsurgical repair and transplantation of OECs or injection of medium without cells. Twenty-one days later the nerves were removed and prepared for either histology or electrophysiological analysis. Footprint analysis was carried out at 7, 14 and 21 days. The OECs survived and integrated into the repaired nerves as indicated by eGFP-expressing cells aligned with neurofilament identified axons bridging the repair site. Moreover, regenerated axons were myelinated by the transplanted OECs and nodes of Ranvier were formed. Conduction velocity in the OEC transplant group was increased in comparison to the microsurgical repair alone, and improved stepping was observed in the transplant group. These results suggest that presentation of OECs at the time of nerve injury enhances regeneration and improves functional outcome. Even a modest improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.