Laurent A. Tchang

Osteoinductivity of engineered cartilaginous templates devitalized by inducible apoptosis

Significance It has been previously reported that hypertrophic cartilage tissues engineered from human mesenchymal stromal cells can efficiently remodel in vivo into bone organs, recapitulating developmental steps of endochondral ossification. We have here demonstrated that the extracellular matrix (ECM) of such engineered cartilage, even in the absence of a living cell component, retains frankly osteoinductive properties. The use of an apoptosis-driven devitalization technique revealed the importance of preserving the ECM integrity and, in particular, the embedded factors to trigger the regenerative process. Although exemplified in a skeletal context, our work outlines the general paradigm of cell-based but cell-free off-the-shelf materials capable of activating endogenous cells toward the formation of specific tissues. The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard “freeze & thaw” (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue.

Tissue decellularization by activation of programmed cell death

Decellularized tissues, native or engineered, are receiving increasing interest in the field of regenerative medicine as scaffolds or implants for tissue and organ repair. The approach, which offers the opportunity to deliver off-the-shelf bioactive materials without immuno-matching requirements, is based on the rationale that extracellular matrix (ECM)-presented cues can be potently instructive towards regeneration. However, existing decellularization protocols typically result in damage to the source ECM and do not allow the controlled preservation of its structural, biochemical and/or biomechanical features. Here we propose the deliberate activation of programmed cell death as a method to selectively target the cellular component of a tissue and thereby to preserve the integrity of the decellularized ECM. In the case of engineered tissues, the approach could be complemented by the use of (i) an immortalized cell line, engineered to undergo apoptosis upon exposure to a chemical inducer, and (ii) a perfusion bioreactor system, supporting efficient removal of cellular material. The combination of these tools may lead to the streamlined development of more appropriate materials, based on engineered and decellularized ECM and including a customized set of signals specifically designed to activate endogenous regenerative processes.

Effects of intersyringe processing on adipose tissue and its cellular components: implications in autologous fat grafting.

Background: Autologous fat grafting is a popular technique in plastic surgery. A mechanical processing method is used to facilitate fat injection. No study has investigated whether this process affects cell quality and preservation of biological functionality. This study analyzed the influence of quick mechanical processing through two interconnected small-diameter syringes (“shuffling”) on both structure and viability of fat tissue, and on viability, clonogenicity, and differentiation of the freshly isolated stromal vascular fraction. Methods: Lipoaspiration was performed in six healthy donors, followed by shuffling the fat either zero, five, or 30 times between two 10-cc syringes. Thereafter, fat was applied through a 1.5-mm cannula as in a clinical setting for autologous fat grafting. Analysis of different treatment conditions was conducted. Immunofluorescent staining allowed assessment of morphology, viability, composition, and damage of the tissue. The stromal vascular fraction was examined for isolation yield, viability, clonogenicity, and differentiation capacity. Results: The process of shuffling changed the macroscopic but not the microscopic structure of the lipoaspirated fat. No difference in cell number, viability, number of lipid droplets, vascular architecture, or ratio of cell composition was found. Analysis of the stromal vascular fraction, apart from large interdonor variability, did not show a significant change in isolation yield, viability, clonogenicity, or adipogenic differentiation capacity of the expanded cells. Conclusions: The mechanical procedure of shuffling lipoaspirated fat does not alter its tissue viability or its microscopic structure. The absence of impact on the stromal vascular fraction in the assessed parameters suggests that shuffling can be executed according to surgical needs.

Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First‐in‐Man Study

Stromal Vascular Fraction (SVF) cells freshly isolated from adipose tissue include osteogenic‐ and vascular‐progenitors, yet their relevance in bone fracture healing is currently unknown. Here, we investigated whether human SVF cells directly contribute to the repair of experimental fractures in nude rats, and explored the feasibility/safety of their clinical use for augmentation of upper arm fractures in elderly individuals. Human SVF cells were loaded onto ceramic granules within fibrin gel and implanted in critical nude rat femoral fractures after locking‐plate osteosynthesis, with cell‐free grafts as control. After 8 weeks, only SVF‐treated fractures did not fail mechanically and displayed formation of ossicles at the repair site, with vascular and bone structures formed by human cells. The same materials combined with autologous SVF cells were then used to treat low‐energy proximal humeral fractures in 8 patients (64‐84 years old) along with standard open reduction and internal fixation. Graft manufacturing and implantation were compatible with intraoperative settings and led to no adverse reactions, thereby verifying feasibility/safety. Biopsies of the repair tissue after up to 12 months, upon plate revision or removal, demonstrated formation of bone ossicles, structurally disconnected and morphologically distinct from osteoconducted bone, suggesting the osteogenic nature of implanted SVF cells. We demonstrate that SVF cells, without expansion or exogenous priming, can spontaneously form bone tissue and vessel structures within a fracture‐microenvironment. The gained clinical insights into the biological functionality of the grafts, combined with their facile, intra‐operative manufacturing modality, warrant further tests of effectiveness in larger, controlled trials. Stem Cells 2016;34:2956–2966

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM‐MSCs) combined with calcium phosphate microparticles and enveloped in a platelet‐rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose‐derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM‐MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM‐MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM‐MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un‐aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM‐MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose‐derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.

Second free radial forearm flap for urethral reconstruction after partial flap necrosis of tube-in-tube phalloplasty with radial forearm flap: A report of two cases

We present a salvage procedure to reconstruct the neo‐urethra after partial flap necrosis occurring in free radial forearm flap (RFF) phalloplasty for sex reassignment surgery. Two cases of tube‐in‐tube phalloplasty using a free sensate RFF are described in which partial flap necrosis occurred involving the complete length of the neo‐urethra and a strip of the outer lining of the neo‐phallus. Neo‐urethra‐reconstruction was performed with a second RFF from the contralateral side providing well‐vascularized tissue. No flap‐related complications were observed. Twelve months postoperatively, both patients were able to void while standing. A satisfactory aesthetic appearance of the neo‐phallus could be preserved with an excellent tactile and erogenous sensitivity. Using this technique, we successfully salvaged the neo‐urethra and reconstructed the outer lining of the neo‐phallus

Power-assisted liposuction (PAL) of multiple symmetric lipomatosis (MSL)—a longitudinal study

BACKGROUND Multiple symmetric lipomatosis (MSL) is a rare condition leading to grotesque disfigurement. Complete removal is almost impossible and recurrences inevitable. The objective of this study was to evaluate powered-assisted liposuction (PAL) for the treatment of MSL. METHODS Magnetic resonance imaging (MRI) was performed before and after surgery for quality assessment. To exclude malignancy incisional biopsies were taken before surgery. Outcome measures included aspiration volumes, duration of surgery, early morbidity, recurrence rates, and overall patient satisfaction. RESULTS Seven male patients aged between 43 and 70 years were identified. The mean liposuction volume equaled 2948±1566 mL, the mean surgery time 74±28 minutes. One hematoma was observed, whereas 1 recurrence rate was noticed after a mean follow-up of 14 months. Malignancy was excluded by histology in 5 patients. Preoperative MRI confirmed the diagnosis with no signs for malignancy and showed an efficient removal of the lesions. After a mean follow-up of 18 months, a high patient satisfaction was achieved. CONCLUSION PAL can serve as an efficient method for the treatment of MSL. It has a significant effect on tightening of the skin, leading to a high patient satisfaction.

Engineered, axially-vascularized osteogenic grafts from human adipose-derived cells to treat avascular necrosis of bone in a rat model

BACKGROUND Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. The standard of care, vascularized bone grafts, is limited by donor site morbidity and restricted availability. The aim of this study was to generate and test engineered, axially vascularized SVF cells-based bone substitutes in a rat model of AVN. METHODS SVF cells were isolated from lipoaspirates and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5days. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8weeks in vivo, bone formation and vascularization were analyzed. RESULTS Newly-formed bone was found in 80% of SVF-seeded scaffolds after 8weeks but not in unseeded controls. Human ALU+cells in the bone structures evidenced a direct contribution of SVF cells to bone formation. A higher density of regenerative, M2 macrophages was observed in SVF-seeded constructs. In both experimental groups, devitalized bone was revitalized by vascularized tissue after 8 weeks. CONCLUSION SVF cells-based osteogenic constructs revitalized fully necrotic bone in a challenging AVN rat model of clinically-relevant size. SVF cells contributed to accelerated initial vascularization, to bone formation and to recruitment of pro-regenerative endogenous cells. STATEMENT OF SIGNIFICANCE Avascular necrosis (AVN) of bone often requires surgical treatment with autologous bone grafts, which is surgically demanding and restricted by significant donor site morbidity and limited availability. This paper describes a de novo engineered axially-vascularized bone graft substitute and tests the potential to revitalize dead bone and provide efficient new bone formation in a rat model. The engineering of an osteogenic/vasculogenic construct of clinically-relevant size with stromal vascular fraction of human adipose, combined to an arteriovenous bundle is described. This construct revitalized and generated new bone tissue. This successful approach proposes a novel paradigm in the treatment of AVN, in which an engineered, vascularized osteogenic graft would be used as a germ to revitalize large volumes of necrotic bone.

Adipose-Derived Stromal Cells from Lipomas: Isolation, Characterisation and Review of the Literature

Objective: The aim of this study was to characterize adipose-derived stromal cells (ADSCs) from patients diagnosed with multiple symmetric lipomatosis (MSL) in order to obtain potentially new insights into the pathophysiology, pathogenesis and treatment of this disease. Methods: Cells from the stromal vascular fraction were analysed by the colony-forming efficiency assay and flow cytometry using standard markers. Moreover, the power of adipogenic plasticity was evaluated. Finally, a literature review was performed from 1982 to 2015 using the US National Institutes of Healths PubMed database. Results: Three European-descent patients diagnosed with either MSL type I or II could be identified for analysis. The resulting mean colony-forming efficiency assay was 14.3 ± 5%. Flow-cytometric analysis of the ADSCs revealed high levels of CD34 (70 ± 9%), CD45 (37 ± 13%) and CD73 (55.8 ± 14%), whereas low levels of CD31 (16.8 ± 14%) and CD105 (5.8 ± 0.7%) were detected. Furthermore, ADSCs showed a strong adipogenic potential, which is in line with the literature review. The stem cell pool in lipoma shows several alterations in biological activities, such as proliferation, apoptosis and stemness. Conclusions: ADSCs from lipoma may be interesting in the application of regenerative medicine. We discuss possible molecular treatment options to regulate their activities at the source of the MSL.

LOP33: Engineering of Axially Vascularized Bone Grafts for the Treatment of Avascular Bone Necrosis

Introduction: Regenerative Peripheral Nerve Interfaces (RPNIs) are neurotized muscle grafts that control prostheses through electromyography (EMG). RPNI signals have not been quantified during phases of voluntary movements. This study: a) characterizes active RPNI signaling compared to background activity and b) defines the reliability and validity of RPNI function during gait phases of rat walking. Material and Methods: Rat groups were: Control (n=3), RPNI (n=3), Denervated (n=3). Bipolar electrodes were implanted onto the soleus muscles in each group. The Control group was left intact. The Denervated group had the tibial nerve transected. For RPNIs, the soleus muscle was freely grafted to the ipsilateral thigh and neurotized by the transected tibial nerve. While walking on a treadmill, rats were videographed and raw EMG signals were simultaneously recorded. Outcome measurements were integrated EMG (iEMG) and iEMG normalized (NiEMG) to stance, swing, or sit gait phase. Results: Majority of EMG activity was observed within the stance phase—70% for Control and 79% for RPNI—as expected for active soleus postural muscles. Stance NiEMG signals were greater than swing NiEMG averages for Control and RPNI groups (Fig 1). The Denervated group stance and swing NiEMG signals were not different without peripheral nerve control. Fidelity of RPNI stance activity (NiEMG signal to background signal) was 5.6 to 1, or double the Control signal fidelity. Correlations between iEMG and stance time for the Control (r=0.74) and RPNI (r=0.76) indicate strong signal reliability (Fig. 2). Conclusion: Measurements of fidelity, reliability, and validity for RPNI signal detection all exceeded normal probability (p<0.05) during voluntary movement.