Spider silk enhanced tissue engineering of cartilage tissue: Approach of a novel bioreactor model using adipose derived stromal cells.

Human cartilage tissue remains a challenge for the development of therapeutic options due to its poor vascularization and reduced regenerative capacities. There are a variety of research approaches dealing with cartilage tissue engineering. In addition to different biomaterials, numerous cell populations have been investigated in bioreactor-supported experimental setups to improve cartilage tissue engineering. The concept of the present study was to investigate spider silk cocoons as scaffold seeded with adipose-derived stromal cells (ASC) in a custom-made bioreactor model using cyclic axial compression to engineer cartilage-like tissue. For chemical induction of differentiation, BMP-7 and TGF-ß2 were added and changes in cell morphology and de-novo tissue formation were investigated using histological staining to verify chondrogenic differentiation. By seeding spider silk cocoons with ASC, a high colonization density and cell proliferation could be achieved. Mechanical induction of differentiation using a newly established bioreactor model led to a more roundish cell phenotype and new extracellular matrix formation, indicating a chondrogenic differentiation. The addition of BMP-7 and TGF-ß2 enhanced the expression of cartilage specific markers in immunohistochemical staining. Overall, the present study can be seen as pilot study and valuable complementation to the published literature.

[Animal bite injuries caused by domestic and wild animals: retrospective analysis and development of a prognostic score]. / Tierbissverletzungen durch heimische und wilde Tiere ­ eine retrospektive Analyse und Erstellung eines prognostischen Scores.

BACKGROUND: The current guidelines on injuries caused by animal bites recommend surgical debridement and antibiotic treatment. Bite wounds generally lead to high-risk infections due to deep skin lesions. However, bite wounds may differ dramatically in terms of depth and involvement of different anatomic structures. A prognostic classification regarding depth or severity is only available for facial injuries in paediatric patients, provided by Lackmann's score. In this study, we assessed prognostic factors and developed a prospective score for general animal bite injuries. PATIENTS AND METHODS: We performed a retrospective single-centre analysis in patients sustaining animal bite injuries from 2007 to 2017. Patients were subdivided according to animal species, location of bite(s), type of treatment and medical care, admission, length of stay, need for revision, and spectrum of microorganisms. We established and verified a rating score ranging from grade I (superficial scratch) to grade Va (deep wound with bone involvement) and grade Vb (deep wound with joint involvement). RESULTS: This study included 567 patients with animal bite injuries, of which 51.7 % were caused by dogs, 39.2 % by cats and 4.1 % by wild animals. 75.7 % of injuries were located in the hands, 12.9 % in the arms, 5.1 % in the face and 5.3 % in other body parts. The most common microorganism was pasteurella, followed by staphylococci and other aerobic strains. Antibiotic treatment comprised Sultamicillin in 75 % of cases, cephalosporins in 10.2 %, Clindamycin in 7.6 % and other antibiotics in 5.1 % of cases.There was a significant correlation (p < 0.001) between animal species and presence of an infection, type of medical care, rating score and spectrum of microorganisms as well as between rating score and need for revision, duration of antibiotic treatment, mode of admission, treatment and medical care, and length of hospital stay. CONCLUSION: The prognostic value of the established rating score was verified. In addition, the study demonstrated that animal species are an important factor influencing treatment. Being effective against most of the identified microorganisms, Sultamicillin should be used as first-line antibiotic treatment.

Influence of glucose and insulin in human adipogenic differentiation models with adipose-derived stem cells.

Autologous fat grafting represents an attractive source for tissue engineering applications in the field of reconstructive medicine. However, in adipogenic differentiation protocols for human adipose-derived stem cells, the concentration of glucose and insulin varies considerably. With the intent to gain maximum tissue augmentation, we focused on the late phase of adipogenesis. In this study, we modified the differentiation protocol for adipose-derived stem cells by prolongation of the induction period and the application highly concentrated glucose and insulin. Human adipose-derived stem cells were isolated from subcutaneous depots and differentiated in a standard induction medium for the first two weeks, followed by two weeks with varying glucose and insulin concentrations. Morphological changes assessed using Oil-Red-O staining were examined for corresponding alterations in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL). Furthermore, glucose and lactate levels in conditioned media were monitored over the period of differentiation. We found high-glucose media increasing the level of lipid accumulation and the size of single droplets whereas insulin significantly showed a dose-dependent negative effect on fat storage. However, whereas high glucose stimulated PPARγ transcription, expression levels in insulin-treated cells remained constant. Results permit assumptions that a high-glucose medium intensifies the degree of differentiation in mature adipocytes providing conditions to promote graft volume while we have identified highly concentrated insulin treatment as an inhibitor of lipid storage in the late adipogenic differentiation.

Multiple Recurrences in Aggressive Forms of Dupuytren's Disease-Can Patients Benefit from Repeated Selective Fasciectomy?

BACKGROUND: In Dupuytren's disease (DD), limited fasciectomy is the mainstay of surgical therapy in patients at risk of contractures and disease recurrences. New minimally invasive treatments such as injection of collagenase clostridium histolyticum have evolved as a common tool for the preliminary treatment of Dupuytren's contractures. However, recurrences and their therapy remain controversial. In this study, we evaluate the benefit of repeated limited fasciectomy in patients with aggressive forms of the disease and multiple recurrences of contractures. METHODS: We evaluated the outcome of 16 patients undergoing limited fasciectomy 3 or more times on a single hand. RESULTS: Postoperatively, 10 of 13 (76.9%) patients were satisfied with the clinical result after the last operation; 10 of 12 (83.3%) patients would choose to have their surgery repeated, if so needed. The mean improvement of proximal interphalangeal joint range of motion was 59.2 degrees (SD 26.8) and 86.2% (SD 19.9). There were no severe complications after treatment within the observed time period. CONCLUSIONS: Our findings demonstrate that patients with recurrence of contractures after multiple previous treatments in aggressive forms of DD can benefit from surgical intervention. In conclusion, repeated limited fasciectomy remains indicated in patients after previous surgeries with DD.

Plastic reconstructive surgery techniques using VRAM or gracilis flaps in order to successfully treat complex urogenital fistulas.

BACKGROUND: Urogenital fistulas are devastating complications occurring after tumors or trauma. Surgical treatment is challenging. Thus, further surgical repair options are needed for treatment of complex fistulas within the pelvic region. METHODS: Twelve patients with urogenital fistulas were surgically treated in our department from 2004 to 2012. These selected cases fulfilled eligibility criteria for continence-preserving surgery - a history of fistula recurrences with ongoing incontinence after receiving at least two surgical attempts. Five VRAM and eight gracilis flaps were used to cover up given tissue defects and to perform functional continence-preserving reconstruction with mean follow-up of 6.3 years. Data were retrospectively reviewed, and standardized survey was performed to evaluate quality of life of all living patients (n = 10). RESULTS: In all cases, final surgical treatment of the given fistulae by VRAM or gracilis flaps could be achieved, with mean operating time of 5:31 h (range: 4:50-6:48 h) for VRAM flap and 3:11 h (range: 2:04-4:42 h) for gracilis flap. Outcome measures were primarily to avoid fistula recurrence after plastic surgical treatment, and secondary quality of life and survival. All patients had their continence preserved, but two patients died during follow-up period. Postoperative assessment revealed the following: VRAM flap patients (n = 3) showed slight incontinence during the follow-up period, whereas continence was restored in all patients with gracilis flap reconstruction (n = 7). Quality-of-life assessment indicated restoration of quality of life in comparison to general population (women > men). Furthermore, key points of the two presented surgical techniques are demonstrated in detail. CONCLUSION: With preformed VRAM or gracilis flaps, complex urogenital fistulas can be successfully eradicated and continence is restored. The main focus should be the recovery of quality of life, which could be successfully regained. Nevertheless, the continence success rate has to be defined on an individual, case-by-case basis. LEVEL OF EVIDENCE: Original work, cohort study level III.

In Vitro Evaluation of Spider Silk Meshes as a Potential Biomaterial for Bladder Reconstruction.

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we investigate the application of spider major ampullate gland-derived dragline silk from the Nephila edulis spider, a natural biomaterial with outstanding mechanical properties and a slow degradation rate, as a potential scaffold for bladder reconstruction by studying the cellular response of primary bladder cells to this biomaterial. We demonstrate that spider silk without any additional biological coating supports adhesion and growth of primary human urothelial cells (HUCs), which are multipotent bladder cells able to differentiate into the various epithelial layers of the bladder. HUCs cultured on spider silk did not show significant changes in the expression of various epithelial-to-mesenchymal transition and fibrosis associated genes, and demonstrated only slight reduction in the expression of adhesion and cellular differentiation genes. Furthermore, flow cytometric analysis showed that most of the silk-exposed HUCs maintain an undifferentiated immunophenotype. These results demonstrate that spider silk from the Nephila edulis spider supports adhesion, survival and growth of HUCs without significantly altering their cellular properties making this type of material a suitable candidate for being tested in pre-clinical models for bladder reconstruction.

Silks as scaffolds for skin reconstruction.

In this short review, we describe the use of high molecular weight proteins produced in the glands of several arthropods-commonly called silks-for the purpose to enhance human skin wound healing. To this end an extensive literature search has been performed, the publications have been categorized concerning silk preparation and application and summarized accordingly: Scaffolds to promote wound healing were prepared by processing the silks in different ways including solubilization of the protein fibers followed by casting or electrospinning. The silk scaffolds were additionally modified by coating or blending with the intention of further functionalization. In several approaches, the scaffolds were also vitalized with skin cells or stem cells. In vitro and in vivo models were implied to test for safety and efficiency. We conclude that silk scaffolds are characterized by an advantageous biocompatibility as well as an impressive versatility rendering them ideally suited for application in wounds. Nevertheless, further investigation is needed to exploit the full capacity of silk in different wound models and to achieve clinical transfer in time.

Adhesion, vitality and osteogenic differentiation capacity of adipose derived stem cells seeded on nitinol nanoparticle coatings.

Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.

Induction of osteogenic differentiation of adipose derived stem cells by microstructured nitinol actuator-mediated mechanical stress.

The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.

Isolation, characterization, differentiation, and application of adipose-derived stem cells.

While bone marrow-derived mesenchymal stem cells are known and have been investigated for a long time, mesenchymal stem cells derived from the adipose tissue were identified as such by Zuk et al. in 2001. However, as subcutaneous fat tissue is a rich source which is much more easily accessible than bone marrow and thus can be reached by less invasive procedures, adipose-derived stem cells have moved into the research spotlight over the last 8 years.Isolation of stromal cell fractions involves centrifugation, digestion, and filtration, resulting in an adherent cell population containing mesenchymal stem cells; these can be subdivided by cell sorting and cultured under common conditions.They seem to have comparable properties to bone marrow-derived mesenchymal stem cells in their differentiation abilities as well as a favorable angiogenic and anti-inflammatory cytokine secretion profile and therefore have become widely used in tissue engineering and clinical regenerative medicine.