Human skin specific long noncoding RNA HOXC13-AS regulates epidermal differentiation by interfering with Golgi-ER retrograde transport.

After a skin injury, keratinocytes switch from a state of homeostasis to one of regeneration leading to the reconstruction of the epidermal barrier. The regulatory mechanism of gene expression underpinning this key switch during human skin wound healing is enigmatic. Long noncoding RNAs (lncRNAs) constitute a new horizon in the understanding of the regulatory programs encoded in the mammalian genome. By comparing the transcriptome of an acute human wound and skin from the same donor as well as keratinocytes isolated from these paired tissue samples, we generated a list of lncRNAs showing changed expression in keratinocytes during wound repair. Our study focused on HOXC13-AS, a recently evolved human lncRNA specifically expressed in epidermal keratinocytes, and we found that its expression was temporally downregulated during wound healing. In line with its enrichment in suprabasal keratinocytes, HOXC13-AS was found to be increasingly expressed during keratinocyte differentiation, but its expression was reduced by EGFR signaling. After HOXC13-AS knockdown or overexpression in human primary keratinocytes undergoing differentiation induced by cell suspension or calcium treatment and in organotypic epidermis, we found that HOXC13-AS promoted keratinocyte differentiation. Moreover, RNA pull-down assays followed by mass spectrometry and RNA immunoprecipitation analysis revealed that mechanistically HOXC13-AS sequestered the coat complex subunit alpha (COPA) protein and interfered with Golgi-to-endoplasmic reticulum (ER) molecular transport, resulting in ER stress and enhanced keratinocyte differentiation. In summary, we identified HOXC13-AS as a crucial regulator of human epidermal differentiation.

Integrative small and long RNA omics analysis of human healing and nonhealing wounds discovers cooperating microRNAs as therapeutic targets.

MicroRNAs (miR), as important epigenetic control factors, reportedly regulate wound repair. However, our insufficient knowledge of clinically relevant miRs hinders their potential therapeutic use. For this, we performed paired small and long RNA-sequencing and integrative omics analysis in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic nonhealing venous ulcers (VUs). On the basis of the findings, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte migration and growth while promoting inflammatory response. By combining miR expression patterns with their specific target gene expression context, we identified miRs highly relevant to VU pathology. Our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity.

Circular RNA Signatures of Human Healing and Nonhealing Wounds.

Venous ulcers (VUs) have complex and obscure pathogenicity, and effective VU therapies are still lacking. Circular RNAs (circRNAs) have emerged as powerful gene regulators with important roles in health and disease. In this study, we used paired total RNA and small RNA sequencing to profile circRNAs, protein-coding mRNAs, and microRNAs expression in a unique collection of clinical samples: healthy skin and acute wounds at inflammatory and proliferative phases and wound-edge VU biopsies. We unravel a dynamically changed expression pattern of circRNAs during human skin repair and their abnormal expression signature in VU, which are presented as a searchable web resource (www.xulandenlab.com/humanwounds-circrna). We analyzed the coexpression relationship between the circRNAs and mRNAs with weighted correlation network analysis and constructed circRNA‒mRNA‒microRNA networks. This allowed us to expose the regulatory networks specific to the inflammatory and proliferative phases of wound repair and VU, the biological processes the circRNAs may regulate, and the circRNAs that could sponge microRNAs in human wounds. Importantly, we found that hsa-CHST15_0003 and hsa-TNFRSF21_0001, two circRNAs upregulated in VU, hampered epidermal keratinocyte migration while promoting proliferation by modulating gene networks underpinning these cellular processes. This study paves the way to decipher the functional significance of circRNAs in tissue repair.

Vaginal reconstruction using a gluteal transposition flap after abdominoperineal excision for anorectal malignancy.

The purpose of this study is to present and evaluate a surgical method using gluteal flap for combined perineal and vaginal reconstruction after abdominoperineal excision (APE) with partial vaginectomy for anorectal malignancy. The method is a two-centre study of consecutive patients undergoing APE including partial vaginectomy for anorectal tumours, with immediate combined perineal and vaginal reconstruction using gluteal flaps. Follow-up data were retrieved via retrospective review of medical records, questionnaires and gynaecological examinations. Some 34 patients fulfilled the inclusion criteria. At the time of follow-up, 14 (78%) of the 18 patients alive responded to questionnaires. Seven (50%) of the survey responders agreed to undergo gynaecological examination. Major flap-specific complications (Clavien-Dindo > 2) were observed in 3 (9%) patients. Among survey responders, 11 (79%) had been sexually active preoperatively of which five (45%) resumed sexual activity postoperatively and three (27%) resumed vaginal intercourse. These three patients had all implemented an active vaginal health promotion strategy postoperatively. Perineo-vaginal reconstruction using gluteal flap after extended APE for anorectal malignancy is feasible. Although comparable to other methods of reconstruction, the rate of perineo-vaginal complications is high and post-operative sexual dysfunction is substantial. Postoperative strategies for vaginal health promotion may improve sexual function after vaginal reconstruction.

Implant Attributes or Patient Characteristics? Factors Affecting Outcome after Breast Augmentation in Transgender Women.

Implant-based breast augmentation is a valuable tool for treatment of gender dysphoria in transgender women. The aim was to assess whether implant attributes, plane selection, and patient characteristics had an impact on the surgical outcome, and to compare these parameters between transgender and cisgender breast augmentations. Methods: A cohort of transgender women who underwent breast augmentation at our department during 2009-2018 were retrospectively studied. The cohort was also compared with a cohort of 12,884 mainly cisgender women registered in the Swedish breast implant registry (BRIMP) during 2014-2019. Results: A total of 143 transgender individuals were included, with a median follow-up of 5.7 years. Complications occurred in 20 patients (14.0%), four patients (2.8%) underwent acute reoperation, and 20 patients (14.0%) had secondary corrections. No differences were seen in complication rates when comparing prepectoral with subpectoral placement (15.1% versus 12.9%; P = 0.81); size, less than 400 mL versus greater than or equal to 400 mL (14.7% versus 13.3%; P = 0.81), or the shape of the implants, round versus anatomic (10.7% versus 22.2%; P = 0.10). In comparison with the cohort from BRIMP, the transgender cohort had more round implants (72.0% versus 60.7%; P < 0.01), larger implants (44.1% had volumes of 400-599 mL, compared with 25.4%; P < 0.0001), and more prepectoral placement (51.0% versus 7.3%; P < 0.0001). The risk of reoperation less than 30 days was 1.2% in BRIMP and 2.8% in the transgender cohort (P = 0.08). Conclusions: In transgender women, implants are often larger, round, and placed prepectoral' compared with cisgender women. Despite these differences, complication rates were equivalent. Implant attributes, surgical techniques, and patient characteristics were not independently associated with the rate of complications.

Single-Cell Analysis Reveals Major Histocompatibility Complex II‒Expressing Keratinocytes in Pressure Ulcers with Worse Healing Outcomes.

Pressure ulcer (PU) is a chronic wound often seen in patients with spinal cord injury and other bed-bound individuals, particularly in the elderly population. Despite its association with high mortality, the pathophysiology of PU remains poorly understood. In this study, we compared single-cell transcriptomic profiles of human epidermal cells from PU wound edges with those from uninjured skin and acute wounds in healthy donors. We identified significant shifts in the cell composition and gene expression patterns in PU. In particular, we found that major histocompatibility complex class II‒expressing keratinocytes were enriched in patients with worse healing outcomes. Furthermore, we showed that the IFN-γ in PU-derived wound fluid could induce major histocompatibility complex II expression in keratinocytes and that these wound fluid‒treated keratinocytes inhibited autologous T-cell activation. In line with this observation, we found that T cells from PUs enriched with major histocompatibility complex II+ keratinocytes produced fewer inflammatory cytokines. Overall, our study provides a high-resolution molecular map of human PU compared with that of acute wounds and intact skin, providing insights into PU pathology and the future development of tailored wound therapy.

Free Flap Surgery Outcome Related to Antithrombotic Treatment Regime: An Analysis of 1000 Cases.

Autologous free tissue transfer is today an integral part of reconstructive plastic surgery, but still lacks generally accepted guidelines regarding antithrombotic agents. We hypothesized that the overuse of antithrombotic agents could be a risk factor for free flap complications and therefore studied a treatment protocol adjustment. METHODS: Consecutive free flaps between 2005 and 2020 at a single center were analyzed for complications in relation to the use of pre- and intraoperative treatment with three different antithrombotic agents. The use of preoperative low molecular weight heparin (LMWH), intraoperative heparin, and dextran were analyzed in relation to outcome variables, thromboembolic events, or reexploration for hematoma. RESULTS: Nine hundred thirty-one patients underwent 1000 microvascular free flaps for breast (n = 487), head and neck (n = 365), and extremity (n = 148) reconstruction. Within the first postoperative week, 44 cases had a thromboembolic event and 58 cases underwent hematoma-related reexploration. In the multivariate analysis, thromboembolic events were associated with extremity reconstruction (P = 0.02) and smoking (P = 0.02). Hematoma-related reexploration was more common with triple antithrombotic therapy compared with all other treatment regimes (P < 0.05). The number of antithrombotic agents used perioperatively was linearly decreased, from three to none, over the elapsed time period (P < 0.001). CONCLUSIONS: Hematoma was the most common reason for reexploration and was further associated with the use of multiple antithrombotic agents. Cessation of triple treatment was associated with less hematomas and further reduction of antithrombotic agents did not result in any increase of thromboembolic events. Evidence-based guidelines are warranted for antithrombotic regimes in standard free flap surgery.

Improved Surgical Outcome with Double Incision and Free Nipple Graft in Gender Confirmation Mastectomy.

BACKGROUND: Mastectomy and chest-wall contouring is the most common gender confirmation surgery. With increasing prevalence of transgender individuals, there is a demand for better surgical outcomes and aesthetic results. Our aim was to evaluate surgical techniques used and assess modifications in gender confirmation mastectomies at Karolinska University hospital in Stockholm, Sweden. METHODS: A retrospective cohort study was performed on 464 patients undergoing gender confirmation mastectomies in our department between 2009 and 2018. Patient demographics, psychiatric comorbidity, surgical method, and outcome were analyzed. Follow-up was at least one year. RESULTS: The most frequently used surgical technique for gender confirmation mastectomies was double incision with free nipple graft (243 patients, 52.4%), followed by periareolar incision (113 patients, 24.4%) and semicircular incision (67 patients, 14.4%). The double incision technique and periareolar technique were associated with 18.9% and 28.3% complications, 3.3% and 12.4% acute reoperations, 28.4% and 65.5% secondary revisions, respectively. The double incision technique increased from being used in 17.8% of all mastectomies during 2009-2013 to 62.9% during 2014-2018, while periareolar incision decreased from 43.0% to 18.5%. CONCLUSIONS: The current study describes a successful transition of surgical technique from periareolar incision to double incision with free nipple graft in gender confirmation mastectomy, leading to significant improvements in the overall outcome with fewer complications, less acute reoperations and less secondary corrections. Hence, we consider the double incision with free nipple graft technique to be the favored technique in the vast majority of cases in female-to-male chest wall contouring.

miR-19a/b and miR-20a Promote Wound Healing by Regulating the Inflammatory Response of Keratinocytes.

Persistent and impaired inflammation impedes tissue healing and is a characteristic of chronic wounds. A better understanding of the mechanisms controlling wound inflammation is needed. In this study, we show that in human wound-edge keratinocytes, the expressions of microRNA (miR)-17, miR-18a, miR-19a, miR-19b, and miR-20a, which all belong to the miR-17∼92 cluster, are upregulated during wound repair. However, their levels are lower in chronic ulcers than in acute wounds at the proliferative phase. Conditional knockout of miR-17∼92 in keratinocytes as well as injection of miR-19a/b and miR-20a antisense inhibitors into wound edges enhanced inflammation and delayed wound closure in mice. In contrast, conditional overexpression of the miR-17∼92 cluster or miR-19b alone in mice keratinocytes accelerated wound closure in vivo. Mechanistically, miR-19a/b and miR-20a decreased TLR3-mediated NF-κB activation by targeting SHCBP1 and SEMA7A, respectively, reducing the production of inflammatory chemokines and cytokines by keratinocytes. Thus, miR-19a/b and miR-20a being crucial regulators of wound inflammation, the lack thereof may contribute to sustained inflammation and impaired healing in chronic wounds. In line with this, we show that a combinatory treatment with miR-19b and miR-20a improved wound healing in a mouse model of type 2 diabetes.

Complications after pressure ulcer surgery - a study of 118 operations in spinal cord injured patients.

Our aim in this study was to examine which factors are associated with post-operative complications after surgery for pressure ulcers in individuals with spinal cord injury. We performed a retrospective cohort study including all spinal cord injured patients undergoing pressure ulcer surgery in our department between 2002 and 2019. Patient demographics and comorbidity were collected from medical records, as were treatment data and information on post-operative complications within 30 days. Assessment of outcomes was performed through t-tests, χ2-tests and ANOVA. Out of 118 operations, 51 (43%) had a post-operative complication of any kind. The vast majority (44 cases, 86% of all complications) had minor complications (Clavien-Dindo grade I or II). Seven patients (6%) had a complication of Clavien-Dindo grade III or higher, requiring return to theatre or ICU care. We found that a higher age, a low serum albumin (<3.5 g/dl), and over or underweight, were associated with an increased risk of complications (p < 0.05). Out of 143 treated ulcers, 132 were evaluated at a follow-up visit one to two months after surgery, and 99 of these (75%) were healed. Surgery of pressure ulcers in patients with spinal cord injury is not a low-risk venture, however, few patients will suffer serious post-operative complications. A majority of treated ulcers will heal shortly post-operatively. A number of risk factors are associated with post-operative complications, which can be of help to guide patient selection in the future.

Human skin long noncoding RNA WAKMAR1 regulates wound healing by enhancing keratinocyte migration.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

WAKMAR2, a Long Noncoding RNA Downregulated in Human Chronic Wounds, Modulates Keratinocyte Motility and Production of Inflammatory Chemokines.

Chronic wounds represent a major and growing health and economic burden worldwide. A better understanding of molecular mechanisms of normal as well as impaired wound healing is needed to develop effective treatment. Herein we studied the potential role of long noncoding RNA LOC100130476 in skin wound repair. LOC100130476 is an RNA polymerase II-encoded polyadenylated transcript present in both cytoplasm and nucleus. We found that its expression was lower in wound-edge keratinocytes of human chronic wounds compared to normal wounds of healthy donors and intact skin. In cultured keratinocytes, LOC100130476 expression was induced by TGF-ß signaling. By reducing LOC100130476 expression with antisense oligos or activating its transcription with CRISPR/Cas9 Synergistic Activation Mediator system, we showed that LOC100130476 restricted the production of inflammatory chemokines by keratinocytes, while enhancing cell migration. In line with this, knockdown of LOC100130476 impaired re-epithelization of human ex vivo wounds. Based on these results, we named LOC100130476 wound and keratinocyte migration-associated long noncoding RNA 2 (WAKMAR2). Moreover, we identified a molecular network that may mediate the biological function of WAKMAR2 in keratinocytes using microarray. In summary, our data suggest that WAKMAR2 is an important regulator of skin wound healing and its deficiency may contribute to the pathogenesis of chronic wounds.

Improved Head and Neck Free Flap Outcome-Effects of a Treatment Protocol Adjustment from Pre- to Postoperative Radiotherapy.

BACKGROUND: The impact of preoperative radiotherapy on microvascular reconstructive surgery outcome has been a subject of debate. However, data are conflicting and often dependent on local treatment protocols. We have studied the effects of radiotherapy in a unique, single-center setting where a treatment protocol change was undertaken from pre- to postoperative radiotherapy administration for microsurgical head and neck reconstructions. METHODS: A cohort study was conducted for 200 consecutive head and neck free flap cases, where 100 were operated on before and 100 after the treatment protocol adjustment in 2006. Only direct cancer reconstructions were included. Complication rates of anastomosis-related (flap necrosis) and flap bed-related (infection, fistula, and wound dehiscence) complications were compared between irradiated and nonirradiated patients. A multivariate analysis was performed to correct for treatment period. RESULTS: One hundred twenty-six patients had received radiotherapy before reconstruction due to cases of cancer recurrence. There were no significant differences in demographic data or risk factors between irradiated and nonirradiated cases. Irradiated cases had a higher rate of both flap loss (9.5% versus 1.4%; P = 0.034) and flap bed-related complications (29% versus 13%; P = 0.014). However, after multivariate analysis, there was only a significant relationship between preoperative irradiation and infection (odds ratio = 2.51; P = 0.033) and fistula formation (odds ratio = 3.13; P = 0.034). CONCLUSIONS: The current single-center study clearly indicates that preoperative radiotherapy is a risk factor for both infection and fistula formation, most likely related to an impaired flap bed. We suggest postoperative radiotherapy administration whenever possible for oncological reasons, otherwise proper antibiotic cover and meticulous flap insetting to prevent radiation-related infection and fistula formation.

Standardizing the freeze-thaw preparation of growth factors from platelet lysate.

BACKGROUND: Over the past decades, the focus on the regenerative properties of platelets (PLTs) has intensified and many PLT-derived growth factors are readily used in medical settings. A general lack of standardization in the preparation of these growth factors remains, however, and this study therefore examines the dynamics of growth factors throughout the freeze-thaw procedure. STUDY DESIGN AND METHODS: Plateletpheresis (PA) and PLT-poor plasma (PPP) samples were collected from 10 healthy donors. PA was lysed to produce PLT lysate (PL) for 1, 3, 5, 10, and 30 freeze-thaw cycles. The resulting growth factor and cytokine concentrations from PPP, PA, and PL of different cycles were analyzed and compared using enzyme-linked immunosorbent assay and multiplex bead assays. RESULTS: PL produced by the freeze-thaw procedure resulted in approximately four- to 10-fold enrichment of transforming growth factor-ß1, epidermal growth factor, PLT-derived growth factor (PDGF)-AB/BB, PLT factor-4, and fibroblast growth factor-2. The increase in concentrations plateaued at Cycles 3 and 5 and in some cases declined with further cycles. The concentrations of insulin-like growth factor-1, hepatocyte growth factor, vascular endothelial growth factor, and bone morphogenetic protein-2 in PL were essentially comparable to those in PPP. CONCLUSION: Using the freeze-thaw method, optimal preparation of PL with regard to the concentration of growth factors was achieved at Cycles 3 to 5. Based on our findings, the clinical significance of using a greater number of cycles is likely limited.

Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units.

BACKGROUND: Platelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units. MATERIALS AND METHODS: Platelet pooled units (n = 10) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-ß isoform 1, IL-1ß, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ. RESULTS: The concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-ß, PDGF-AB/BB, and PF4 showed an increase of 27.2% (p < 0.0001), 29.5% (p = 0.04) and 8.2% (p = 0.0004), respectively. A decrease was seen in the levels of IGF-1 and FGF-2 with 22% (p = 0.041) and 11% (p = 0.01), respectively. Cytokines were present only in very low concentrations and all other growth factors remained stable with time in storage. CONCLUSION: The composition of mediators in platelet lysate obtained from pathogen-inactivated platelet units differs when produced from fresh and stored platelet units, respectively. This underscores the need for further standardization and optimization of this important product, which potentially may influence the clinical effects.

Effects of a formalized collaboration between plastic and orthopedic surgeons in severe extremity trauma patients; a retrospective study.

BACKGROUND: Severe trauma to the extremities often includes a combination of fractures and soft tissue injuries. Several publications support that the patient outcome is better when skeletal stabilization is followed by early soft-tissue coverage. In an effort to optimize the treatment of these patients, we established a formalized collaboration in 2008 between the Departments of reconstructive plastic surgery and orthopedics at the Karolinska University Hospital. METHODS: A retrospective review was conducted for all patients who had suffered severe extremity trauma and received either a free or a pedicled flap for extremity reconstruction. We compared the management of patients 0-4 years before and 0-4 years after the collaboration started especially with respect to; choice of flap, time to flap coverage, number of operations/revisions, total in-hospital stay. RESULTS: After initiation of the collaboration, the number of flaps increased from 13 flaps (5 free and 8 pedicled) to 44 flaps (21 free and 23 pedicled). Fewer postoperative revisions was seen, as well as shorter in-hospital stay. CONCLUSIONS: The present study highlights the importance of formalized collaboration between orthopedic and plastic surgeons in severe extremity trauma patients. The concept of an interdisciplinary approach has led to an increased number of trauma patients referred for plastic surgical consultation, an increased number of flaps, fewer postoperative revisions and shorter hospital stay.

Lower extremity soft tissue reconstruction and amputation rates in patients with open tibial fractures in Sweden during 1998-2010.

BACKGROUND: The rates of soft tissue reconstruction and amputation after open tibial fractures have not been studied on a national perspective. We aimed to determine the frequency of soft tissue coverage after open tibial fracture as well as primary and secondary amputation rates. METHODS: Data on all patients (> = 15 years) admitted to hospital with open tibial fractures were extracted from the Swedish National Patient Register (1998-2010). All surgical procedures, re-admissions, and mechanisms of injury were analysed accordingly. The risk of amputation was calculated using logistic regression (adjusted for age, sex, mechanism of injury, reconstructive surgery and fixation method). The mean follow-up time was 6 (SD 3.8) years. RESULTS: Of 3,777 patients, 342 patients underwent soft tissue reconstructive surgery. In total, there were 125 amputations. Among patients with no reconstructive surgery, 2% (n = 68 patients) underwent amputation. In an adjusted analysis, patients older than 70 years (OR = 2.7, 95%, CI = 1.1-6) and those who underwent reconstructive surgery (OR = 3.1, 95% CI = 1.6-5.8) showed higher risk for amputation. Fixations other than intramedullary nailing (plate, external fixation, closed reduction and combination) as the only method were associated with a significant higher risk for amputation (OR 5.1-14.4). Reconstruction within 72 hours (3 days) showed better results than reconstruction between 4-90 days (p = 0.04). CONCLUSIONS: The rate of amputations after open tibial fractures is low (3.6%). There is a higher risk for amputations with age above 70 (in contrast: male sex and tissue reconstruction are rather indicators for more severe soft tissue injuries). Only a small proportion of open tibial fractures need soft tissue reconstructive surgery. Reconstruction with free or pedicled flap should be performed within 72 hours whenever possible.

Osteogenically-induced human dermal fibroblasts as a tool to regenerate bone.

Engineering of bone tissue could help to overcome the need for extensive reconstruction and associated donor site morbidity, and it has been proposed that osteogenic biomaterials, which are scaffolds that contain osteocompetent cells, could be used to fill large bone defects. This study investigated the potential of osteogenically-induced human dermal fibroblasts cultured on gelatin microcarriers combined with platelet-rich plasma in a model of a femoral defect in athymic rats. Defects were transplanted with one of the following six combinations: 1 = sodium chloride, 2 = platelet-rich plasma, 3 = microcarriers + platelet-rich plasma, 4 = human dermal fibroblasts on microcarriers + platelet-rich plasma, 5 = human osteoblasts on microcarriers + platelet-rich plasma, and 6 = osteogenically induced human dermal fibroblasts on microcarriers + platelet-rich plasma. The femoral defects were assessed 4 weeks postoperatively with computed tomography (CT), routine histological staining, fluorescence in situ hybridisation, and polyclonal antibodies directed towards osteocalcin and osteonectin. Radiographs of all groups taken 4 weeks postoperatively showed unhealed defects. Femoral defects transplanted with osteogenically-induced human dermal fibroblasts on microcarriers (group 6) contained dense clusters of cells with large quantities of extracellular matrix. These clusters were exclusive to this group and stained strongly for osteocalcin and osteonectin. Fluorescence in situ hybridisation showed viable human cells in femoral defects that had been transplanted with microcarriers seeded with cells, which confirmed the survival of implanted cells. In conclusion, osteogenically-induced human dermal fibroblasts survived in this new niche, and bone-like structures were apparent in the defects.

Interpreted gene expression of human dermal fibroblasts after adipo-, chondro- and osteogenic phenotype shifts.

Autologous cell-based therapies promise important developments for reconstructive surgery. In vitro expansion as well as differentiation strategies could provide a substantial benefit to cellular therapies. Human dermal fibroblasts, considered ubiquitous connective tissue cells, can be coaxed towards different cellular fates, are readily available and may altogether be a suitable cell source for tissue engineering strategies. Global gene expression analysis was performed to investigate the changes of the fibroblast phenotype after four-week inductions toward adipocytic, osteoblastic and chondrocytic lineages. Differential gene regulation, interpreted through Gene Set Enrichment Analysis, highlight important similarities and differences of induced fibroblasts compared to control cultures of human fibroblasts, adipocytes, osteoblasts and articular chondrocytes. Fibroblasts show an inherent degree of phenotype plasticity that can be controlled to obtain cells supportive of multiple tissue types.

Engineering three-dimensional cartilage- and bone-like tissues using human dermal fibroblasts and macroporous gelatine microcarriers.

The creation of tissue-engineered cartilage and bone, using cells from an easily available source seeded on a suitable biomaterial, may have a vast impact on regenerative medicine. While various types of adult stem cells have shown promising results, their use is accompanied by difficulties associated with harvest and culture. The proposed inherent plasticity of dermally derived human fibroblasts may render them useful in tissue-engineering applications. In the present study, human dermal fibroblasts cultured on macroporous gelatine microcarriers encapsulated in platelet-rich plasma into three-dimensional constructs were differentiated towards chondrogenic and osteogenic phenotypes using specific induction media. The effect of flow-induced shear stress on osteogenic differentiation of fibroblasts was also evaluated. The generated tissue constructs were analysed after 4, 8 and 12 weeks using routine and immunohistochemical stainings as well as an enzyme activity assay. The chondrogenic-induced tissue constructs were composed of glycosaminoglycan-rich extracellular matrix, which stained positive for aggrecan. The osteogenic-induced tissue constructs were composed of mineralised extracellular matrix containing osteocalcin and osteonectin, with cells showing an increased alkaline phosphatase activity. Increased osteogenic differentiation was seen when applying flow-induced shear stress to the culture. Un-induced fibroblast controls did not form cartilage- or bone-like tissues. Our findings suggest that primary human dermal fibroblasts can be used to form cartilage- and bone-like tissues in vitro when cultured in specific induction media.