Jian Farhadi

Cell differentiation by mechanical stress

Growth factors, hormones, and other regulatory molecules are traditionally required in tissue engineering studies to direct the differentiation of progenitor cells along specific lineages. We demonstrate that mechanical stimulation in vitro, without ligament‐selective exogenous growth and differentiation factors, induces the differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages). A bioreactor was designed to permit the controlled application of ligament‐like multidimensional mechanical strains (translational and rotational strain) to the undifferentiated cells embedded in a collagen gel. The application of mechanical stress over a period of 21 days up‐regulated ligament fibroblast markers, including collagen types I and III and tenascin‐C, fostered statistically significant cell alignment and density and resulted in the formation of oriented collagen fibers, all features characteristic of ligament cells. At the same time, no up‐regulation of bone or cartilage‐specific cell markers was observed.

Three‐Dimensional Perfusion Culture of Human Adipose Tissue‐Derived Endothelial and Osteoblastic Progenitors Generates Osteogenic Constructs with Intrinsic Vascularization Capacity

In this study, we aimed at generating osteogenic and vasculogenic constructs starting from the stromal vascular fraction (SVF) of human adipose tissue as a single cell source. SVF cells from human lipoaspirates were seeded and cultured for 5 days in porous hydroxyapatite scaffolds by alternate perfusion through the scaffold pores, eliminating standard monolayer (two‐dimensional [2D]) culture. The resulting cell‐scaffold constructs were either enzymatically treated to extract and characterize the cells or subcutaneously implanted in nude mice for 8 weeks to assess the capacity to form bone tissue and blood vessels. SVF cells were also expanded in 2D culture for 5 days and statically loaded in the scaffolds. The SVF yielded 5.9 ± 3.5 × 105 cells per milliliter of lipoaspirate containing both mesenchymal progenitors (5.2% ± 0.9% fibroblastic colony forming units) and endothelial‐lineage cells (54% ± 6% CD34+/CD31+ cells). After 5 days, the total cell number was 1.8‐fold higher in 2D than in three‐dimensional (3D) cultures, but the percentage of mesenchymal‐ and endothelial‐lineage cells was similar (i.e., 65%–72% of CD90+ cells and 7%–9% of CD34+/CD31+ cells). After implantation, constructs from both conditions contained blood vessels stained for human CD31 and CD34, functionally connected to the host vasculature. Importantly, constructs generated under 3D perfusion, and not those based on 2D‐expanded cells, reproducibly formed bone tissue. In conclusion, direct perfusion of human adipose‐derived cells through ceramic scaffolds establishes a 3D culture system for osteoprogenitor and endothelial cells and generates osteogenic‐vasculogenic constructs. It remains to be tested whether the presence of endothelial cells accelerates construct vascularization and could thereby enhance implanted cell survival in larger size implants.

Cell yield, proliferation, and postexpansion differentiation capacity of human ear, nasal, and rib chondrocytes.

Human ear, nasal, and rib chondrocytes were compared with respect to their suitability to generate autologous cartilage grafts for nonarticular reconstructive surgery. Cells were expanded for two passages in medium containing 10% fetal bovine serum without (control) or with transforming growth factor beta(1) (TGF-beta(1)), fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor bb (PDGF-bb) (TFP). Expanded cells were cultured as three-dimensional pellets in chondrogenic serum-free medium containing insulin, dexamethasone, and TGF-beta(1). Chondrocytes from all three sources were successfully isolated, increased their proliferation rate in response to TFP, and dedifferentiated during passaging. Redifferentiation by ear and nasal, but not rib, chondrocytes was enhanced after TFP expansion, as assessed by the significant increase in glycosaminoglycan (GAG)/DNA content and collagen type II mRNA expression in the resulting pellets. TFP-expanded ear and nasal chondrocytes generated pellets of better quality than rib chondrocytes, as assessed by the significantly higher GAG/DNA content and collagen type II mRNA expression, and by the relative stain intensities for GAG and collagen types I and II. In conclusion, postexpansion cell yields suggest that all three sources investigated could be used to generate autologous grafts of clinically relevant size. However, ear and nasal chondrocytes, if expanded with TFP, display superior postexpansion chondrogenic potential and may be a preferred cell source for cartilage tissue engineering.

Factors affecting post-operative complications following skin sparing mastectomy with immediate breast reconstruction

UNLABELLEDnSkin sparing mastectomy (SSM) followed by immediate breast reconstruction (IBR) is not only oncologically safe but provides also significant benefits both cosmetically and functionally. The superiority of this technique can only be fully established, however, by developing a framework for minimising complications. The present study seeks to elucidate the key factors affecting outcome.nnnMETHODSnData for all skin sparing mastectomies with immediate autologous and implant based reconstructions, performed in a three year period (2006-2008) was retrospectively collated. Complications were classified into major and minor. Patients were excluded who had flap loss due to vascular complications.nnnRESULTSnThe total number analysed was 151. 17.2% had major complications, 23% had minor and 61% had no complications. The Wise and the tennis incision had significantly higher rates of wound dehiscence when compared with the periareolar incision (pxa0=xa00.025, pxa0=xa00.098). There was no significant difference between diathermy or blade dissection techniques, or the use of subcutaneous adrenaline infiltration. Increasing BMI was associated with increased skin flap necrosis and wound dehiscence, and an excised breast mass of greater than 750xa0g and a sternal notch to nipple length of greater than 26xa0cm are associated as well with increased flap-related complications (pxa0=xa00.0002, pxa0=xa00.0049).nnnCONCLUSIONnFactors such as Wise pattern and tennis racquet incision, BMI and breast mass and sternal notch to nipple length adversely affect skin sparing mastectomy flap morbidity. These factors should be factored in to patient selection and operative planning especially for obese and large breasted women undergoing skin sparing mastectomy with immediate breast reconstruction.

Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial.

BACKGROUNDnAutologous native cartilage from the nasal septum, ear, or rib is the standard material for surgical reconstruction of the nasal alar lobule after two-layer excision of non-melanoma skin cancer. We assessed whether engineered autologous cartilage grafts allow safe and functional alar lobule restoration.nnnMETHODSnIn a first-in-human trial, we recruited five patients at the University Hospital Basel (Basel, Switzerland). To be eligible, patients had to be aged at least 18 years and have a two-layer defect (≥50% size of alar subunit) after excision of non-melanoma skin cancer on the alar lobule. Chondrocytes (isolated from a 6 mm cartilage biopsy sample from the nasal septum harvested under local anaesthesia during collection of tumour biopsy sample) were expanded, seeded, and cultured with autologous serum onto collagen type I and type III membranes in the course of 4 weeks. The resulting engineered cartilage grafts (25 mmu2008×u200825 mmu2008×u20082 mm) were shaped intra-operatively and implanted after tumour excision under paramedian forehead or nasolabial flaps, as in standard reconstruction with native cartilage. During flap refinement after 6 months, we took biopsy samples of repair tissues and histologically analysed them. The primary outcomes were safety and feasibility of the procedure, assessed 12 months after reconstruction. At least 1 year after implantation, when reconstruction is typically stabilised, we assessed patient satisfaction and functional outcomes (alar cutaneous sensibility, structural stability, and respiratory flow rate).nnnFINDINGSnBetween Dec 13, 2010, and Feb 6, 2012, we enrolled two women and three men aged 76-88 years. All engineered grafts contained a mixed hyaline and fibrous cartilage matrix. 6 months after implantation, reconstructed tissues displayed fibromuscular fatty structures typical of the alar lobule. After 1 year, all patients were satisfied with the aesthetic and functional outcomes and no adverse events had been recorded. Cutaneous sensibility and structural stability of the reconstructed area were clinically satisfactory, with adequate respiratory function.nnnINTERPRETATIONnAutologous nasal cartilage tissues can be engineered and clinically used for functional restoration of alar lobules. Engineered cartilage should now be assessed for other challenging facial reconstructions.nnnFUNDINGnFoundation of the Department of Surgery, University Hospital Basel; and Krebsliga beider Basel.

Preoperative Immunonutrition Suppresses Perioperative Inflammatory Response in Patients with Major Abdominal Surgery—A Randomized Controlled Pilot Study

Background/AimPerioperative administration of immunoenriched diets attenuates the perioperative inflammatory response and reduces postoperative infection complications. However, many questions still remain unresolved in this area, such as the length of diet administration, diet composition, and the mechanisms involved. We performed an open, randomized, triple-arm study comparing the effect of two perioperative feeding regimens with a postoperative one.Methods46 candidates for major elective surgery for malignancy in the upper gastrointestinal tract were randomized to drink preoperatively either 1 L of an immunoenriched formula (Impact) for 5 days (IEF group) or 1 L of Impact plus (Impact enriched with glycine) for 2 days (IEF plus group). The same product as the patient received preoperatively was given to both groups for 7 days postoperatively. In the control group (CON group), patients only received Impact for 7 days postoperatively; there was no preoperative treatment. The main outcome measures were postoperative C-reactive protein (CRP) serum levels.ResultsIn the two preoperatively supplemented groups (treatment groups), perioperative endotoxin levels, CRP (postoperative day 7), and TNF-α (postoperative days 1 and 3) levels were significantly lower compared to the CON group (p < .01). Furthermore, the length of postoperative IMU/ICU stay (Impact 1.9 ± 1.3 days; Impact plus 2.2 ± 1.1 days; control group 5.9 ± 0.8 days) and length of hospital stay (Impact 19.7 ± 2.3 days; Impact plus 20.1 ± 1.3 days; control group 29.1 ± 3.6 days) were both reduced in the treatment groups compared to the control group. Infectious complications (Impact 2/14 (14%); Impact plus 5/17 (29%); control group 10/15 (67%)) also showed a trend toward reduction in the treatment groups.ConclusionsPerioperative administration of an immunoenriched diet significantly reduces systemic perioperative inflammation and postoperative complications in patients undergoing major abdominal cancer surgery, when compared with postoperative diet administration alone. A shortened preoperative feeding regimen of 2 days with Impact enriched with glycine (Impact plus) was as effective as Impact administered for 5 days preoperatively.

In vitro osteogenic differentiation and in vivo bone-forming capacity of human isogenic jaw periosteal cells and bone marrow stromal cells.

Objective:To compare the in vitro osteogenic differentiation and in vivo ectopic bone forming capacity of human bone marrow stromal cells (BMSCs) and jaw periosteal cells (JPCs), and to identify molecular predictors of their osteogenic capacity. Summary Background Data:JPC could be an appealing alternative to BMSC for the engineering of cell-based osteoinductive grafts because of the relatively easy access to tissue with minimal morbidity. However, the extent of osteogenic capacity of JPC has not yet been established or compared with that of BMSC. Methods:BMSCs and JPCs from the same donors (N = 9), expanded for 2 passages, were cultured for 3 weeks in osteogenic medium either in monolayers (Model I) or within 3-dimensional porous ceramic scaffolds, following embedding in fibrin gel (Model II). Cell-fibrin-ceramic constructs were also implanted ectopically in nude mice for 8 weeks (Model III). Cell differentiation in vitro was assessed biochemically and by real-time RT-PCR. Bone formation in vivo was quantified by computerized histomorphometry. Results:JPCs had lower alkaline phosphatase activity, deposited smaller amounts of calcium (Model I), and expressed lower mRNA levels of bone sialoprotein, osteopontin, and osterix (Models I and II) than BMSCs. JPCs produced ectopic bone tissue at lower frequency and amounts (Model III) than BMSCs. Bone sialoprotein, osteopontin, and osterix mRNA levels by BMSCs or JPCs in Model II were markedly higher than in Model I and significantly more expressed by cells that generated bone tissue in Model III. Conclusions:Our data indicate that JPCs, although displaying features of osteogenic cells, would not be as reliable as BMSCs for cell-based bone tissue engineering, and suggest that expression of osteoblast-related markers in vitro could be used to predict whether cells would be osteoinductive in vivo.

Free fibula donor-site morbidity: clinical and biomechanical analysis.

The aim of this study was the quantitative assessment of donor-site morbidity after fibula harvest. Ten patients were identified with a unilateral fibula harvesting and healthy contralateral side. The average follow-up time was 32.3 months. The study design included standardized questionnaire; morbidity point-evaluation system; orthopedic-biomechanical evaluation, including true ankle motion, clinical-functional American Orthopedic Foot and Ankle Society (AOFAS) hindfoot score, pedobarography, measurement of maximal voluntary isometric strength, and radiologic examination using an ankle osteoarthritis grading. Average donor-side morbidity score was low, with 2.6 points (range, 0 to 13). Average AOFAS hindfoot score was good, with 87.3 points (range, 74 to 100). Pedobarography showed significant decrease of the big toe pushup force. Isometric strength measurement showed significant reduction of the peroneus longus muscle. Discrete to mild medial ankle osteoarthritis was found in 70% of the cases. The overall donor-side morbidity was low, but an eversion torque deficit could initiate or support a progressive functional ankle instability and in long-term cause or accelerate a ligamentous secondary ankle osteoarthritis.

Immediate breast reconstruction with acellular dermal matrix: Factors affecting outcome

BACKGROUNDnThe use of acellular dermal matrix (ADM) for coverage of the lower pole in immediate implant-based breast reconstruction has changed surgeons practice. We present our experience using a porcine ADM (Strattice), focusing on short-term outcomes, patient selection, and technique adaptations that may influence outcome.nnnMETHODSnA two-center, retrospective, cohort study was performed from December 2008 to October 2012 at Guys and St. Thomas Hospitals, London, and Clinic Pyramide, Zürich. The study period was divided into two periods: Period 1 which spanned from December 2008 to October 2010 and Period 2 from January 2011 to October 2012 wherein technique adaptations were introduced. Short-term complications after reconstructive surgery were compared between Periods 1 and 2.nnnRESULTSnA total of 149 patients underwent 200 reconstructions (110 one-stage and 90 two-stage) following oncologic (134 breasts) or prophylactic (66 breasts) mastectomy. The mean follow-up was 22.2 months. The total complication rate was 32.5%, including infection, 11.5%; hematoma, 5%; seroma, 10.5%; skin necrosis, 3.5%; and serious wound breakdowns with implant exposure, 1.5%. Complications resulted in 3% requiring an early exchange of implant/expander and in 12.5% requiring explantation. A significant reduction in total complications, infection, implant exposure, and implant loss were noted in Period 2. Multivariate analysis showed time period of surgery (Period 1), single-stage reconstruction, and patient characteristics (mastectomy weight>600 g, or body mass index (BMI)>30, or smoking) to be statistically significant risk factors for the development of postoperative complications. Neoadjuvant chemotherapy showed a trend towards higher complication rates.nnnCONCLUSIONnThe high rate of early complications in this study was mostly related to patient characteristics and learning curves and highlights the importance of patient selection and technique principles in optimizing the outcome.

Anatomical basis and clinical application of the infragluteal perforator flap.

Background: When selecting flaps for coverage of pressure ulcers of the sacrum and perineal region in paraplegic patients, long-term high recurrence rates should be considered. Therefore, the authors developed an infragluteal perforator flap to avoid “burning bridges” for future reconstruction. Methods: Infragluteal perforator flaps were dissected in five fresh human cadavers to define the anatomy of the cutaneous branches of the descending branch of the inferior gluteal artery and cluneal nerves and define anatomical landmarks for clinical application. In a series of 13 paraplegic patients, the authors used perforator-based flaps (additional skin bridge) to cover four perineal ulcers and one sacral ulcer and perforator flaps to cover six perineal and two sacral ulcers. Donor sites were closed by direct approximation. Results: Twelve of 13 flaps healed uneventfully. In all cadaver and clinical dissections, one or two cutaneous branches of the descending branch of the inferior gluteal artery and one or two cluneal nerves were found at the lower border of the gluteus maximus muscle supplying the infragluteal perforator flap. These direct cutaneous branches allowed dissection of inferior gluteal perforator flaps with improved flap mobility compared with the perforator-based flaps. The descending branch of the inferior gluteal artery could always be spared for future flaps. Conclusions: The infragluteal perforator flap is a versatile and reliable flap for coverage of ischial and sacral pressure sores. It can be designed as a perforator-based or perforator flap and could provide a sensate flap in ambulatory patients. Donor-site morbidity is minimal, and options for future flaps of the gluteal and posterior thigh region are preserved.