BACKGROUNDOsteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells (BMSCs) in the repair of a large, high-load-bearing, osteochondral defect in a canine model.

METHODSThe biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects (4.2 mm (diameter) × 6 mm (depth)) were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold (control group). The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation.

RESULTSThe osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group (6 months) was (6.95 ± 0.79) N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was (158.16± 24.30) N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was (218 ± 21.6) µg/mg (dry weight), 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, with no significant difference between the experimental and control groups.

CONCLUSIONThe extracellular matrix-derived, integrated, biphasic scaffold shows potential for the repair of large, high-load-bearing osteochondral defects.

Animals ; Bone Marrow Cells ; cytology ; Bone Regeneration ; physiology ; Cartilage, Articular ; surgery ; Dogs ; Extracellular Matrix ; chemistry ; Mesenchymal Stromal Cells ; cytology ; ultrastructure ; Microscopy, Electron, Scanning ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry ; X-Ray Microtomography

BACKGROUNDCartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage. We had previously developed a natural, human cartilage extracellular matrix (ECM)-derived scaffold for in vivo cartilage tissue engineering in nude mice. However, before these scaffolds can be used in clinical applications in vivo, the in vitro effects should be further explored.

METHODSWe produced cartilage in vitro using a natural cartilage ECM-derived scaffold. The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy (SEM), micro-computed tomography (micro-CT), histological staining, cytotoxicity assay, biochemical and biomechanical analysis. After being chondrogenically induced, the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry. The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining. Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.

RESULTSSEM and micro-CT revealed a 3-D interconnected porous structure. The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris, and stained positive for safranin O and collagen II. Viability staining indicated no cytotoxic effects of the scaffold. Biochemical analysis showed that collagen content was (708.2-44.7) µg/mg, with GAG (254.7 ± 25.9) µg/mg. Mechanical testing showed the compression moduli (E) were (1.226 ± 0.288) and (0.052 ± 0.007) MPa in dry and wet conditions, respectively. Isolated canine bone marrow-derived stem cells (BMSCs) were induced down a chondrogenic pathway, labeled with PKH26, and seeded onto the scaffold. Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM. The cell-scaffold constructs contained pink, smooth and translucent cartilage-like tissue after 3 weeks of culture. We observed evenly distributed cartilage ECM proteoglycans and collagen type II around seeded BMSCs on the surface and inside the pores throughout the scaffold.

CONCLUSIONThis study suggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.

Animals ; Biomechanical Phenomena ; Cartilage ; cytology ; Cell Survival ; Cells, Cultured ; Dogs ; Extracellular Matrix ; physiology ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stromal Cells ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVE: To investigate the risk factors for cow's milk protein allergy (CMPA) among infants through a multicenter clinical study. METHODS: A total of 1 829 infants, aged 1-12 months, who attended the outpatient service of the pediatric department in six hospitals in Shenzhen, China from June 2016 to May 2017 were enrolled as subjects. A questionnaire survey was performed to screen out suspected cases of CMPA. Food avoidance and oral food challenge tests were used to make a confirmed diagnosis of CMPA CMPA. A multivariate logistic regression analysis was used to investigate the risk factors for CMPA. RESULTS: Among the 1 829 infants, 82 (4.48%) were diagnosed with CMPA. The multivariate logistic regression analysis showed that maternal food allergy (OR=4.91, 95%CI: 2.24-10.76, P<0.05), antibiotic exposure during pregnancy (OR=3.18, 95%CI: 1.32-7.65, P<0.05), and the introduction of complementary food at an age of <4 months (OR=3.55, 95%CI: 1.52-8.27, P<0.05) were risk factors for CMPA, while exclusive breastfeeding (OR=0.21, 95%CI: 0.08-0.58, P<0.05) and the introduction of complementary food at an age of >6 months (OR=0.38, 95%CI: 0.17-0.86, P<0.05) were protective factors. CONCLUSIONS The introduction of complementary food at an age of <4 months, maternal food allergy, and antibiotic exposure during pregnancy are risk factors for CMPA in infants.
Animals ; Cattle ; China ; Female ; Humans ; Infant ; Milk Hypersensitivity ; Milk Proteins ; Pregnancy ; Risk Factors ; Surveys and Questionnaires