Background: Recombinant human bone morphogenetic proteins (rhBMPs) have been characterized especially chondrogenic and osteogenic activity both in vitro and in vivo studies. However, delivery of more than one growth factor by sustained release carrier to orthopedic site has yet been questionable in terms of efficacy and synergism.
Objective: Evaluate osteoinductivity and synergistic effect of rhBMP-2 and -7 using absorbable collagen sponge (ACS) carrier system in vivo.
Methods: cDNA of BMP-2 and -7 active domains were cloned and expressed in Escherichia coli BL21 StarTM (DE3) using pRSETc expression system. Then, the purified rhBMPs were loaded onto ACS and evaluated by in vivo rat subcutaneous bioassay. Two and eight weeks postoperatively, all treated groups were histologically verified for evidence of new bone formation and neovascularization by hematoxylin-eosin staining and light microscopy.
Results: The Wistar rat treated with rhBMP-2 or -7/ACS exhibited new bone formation, compared to ACS control. The group treated with ACS supplemented with both rhBMP-2 and -7 significantly showed the osteoid matrix very well-organized into trabeculae-like structure with significant blood vessel invasion.
Conclusion: The osteogenic induction of rhBMPs was combined with ACS carrier in the in vivo bioassay. In addition, the combination of both two potent recombinant osteoinductive cytokines, rhBMP-2 and -7, with ACS carrier demonstrated synergistic effect and might be a more promising and effective choice for therapeutic applications.
Background: Stem cell factor is a pleiotropic cytokine produced by several cell types including fibroblasts, bone marrow stromal cells, mast cells, and endothelial cells. In addition, stem cell factor is an important hematopoietic growth factor, which binds to and activates the ligand for the tyrosine kinase-type receptor c-kit. Objectives: Analyze concentration of stem cell factor within gingival crevicular fluid (GCF) in both periodontal health and disease and to determine the correlation of stem cell factor in GCF and inflammatory status of periodontal tissues. Materials and methods: Forty-five subjects (aged 24 to 75 years) were classified into the following three groups according to their periodontal tissue status as group I (clinically healthy gingiva with no loss of attachment), group II (gingivitis with no attachment loss), and group III (periodontitis). GCF samples collected from each patient were examined for stem cell factor level using enzyme-linked immunosorbant assay. Results: The maximum level of stem cell factor in GCF was obtained for group III (71.8±7.8 pg/g protein), and the lowest mean stem cell factor concentration in GCF was observed for group I (22.1±7.3 pg/g protein). The GCF stem cell factor level of patients in group III was statistically higher than that in group II (p <0.04) and group I (p <0.001). In addition, the mean GCF levels of stem cell factor in group II (48.1±7.5 pg/g protein) were significantly higher than those in group I (p <0.02). There was a positive correlation between stem cell factor in GCF and gingival inflammation index (r=0.59, p <0.001) Conclusion: GCF levels of stem cell factor increased in parallel with the severity of periodontal disease. Its levels in GCF could be potentially useful as a biochemical marker of periodontal inflammation and the host response.
Thakoon Thitiset, Supranee Buranapraditkul, Siriporn Damrongsakkul and Sittisak Honsawek
Background: Cell-based therapy has achieved good functional recovery for tissue repair. Mesenchymal stem cells (MSCs) exhibit multilineage potential, long-term viability, and capacity for self-renewal. Periosteum-derived mesenchymal stem cells (PD cells) may be an attractive cell source for tissue engineering because of their easy accessibility and reduced ethical concerns.
Objectives: To isolate and investigate the phenotypic and functional characteristics of mesenchymal stem cells derived from human periosteum. We also examined the differentiation of PD cells with a trilineage differentiation assay to determine whether they were MSCs.
Materials and Methods: Periosteum-derived cells were cultured in osteogenic, chondrogenic or adipogenic media to evaluate their multilineage differentiation potential. Adherent fibroblast-like cells were analyzed by flow cytometry for MSC cell surface markers. Differentiation of PD cells into osteogenic, chondrogenic, and adipogenic lineages was also evaluated by von Kossa, Alizarin red, Alcian blue, and oil red O stains, respectively. Expression of mesenchymal stem cell markers were assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
Results: We successfully isolated and expanded MSCs from human periosteum. Flow cytometry revealed that PD cells were positive for mesenchymal adhesion cell markers (CD29, CD44, CD90, and CD105) and negative for hematopoietic markers (CD34 and CD45). In osteogenic differentiation, calcium accumulation (positive von Kossa and Alizarin red) and RUNX2, alkaline phosphatase, collagen type I, osteopontin genes were detected. In adipogenic differentiation, the cells displayed oil red O positive and expressed lipoprotein lipase and peroxisome proliferator-activated receptor-gamma (PPAR-γ) associated with adipogenesis. The cells grown in chondrogenic conditions were positively stained for Alcian blue and expressed SOX-9.
Conclusion: PD cells presented osteogenic, chondrogenic, and adipogenic differentiation abilities in vitro and could provide an alternative cellular source for tissue repair in clinical applications.
Jariya Poonpukdee, Chookiet Chalermpanpipat, Sunchai Payungporn and Sittisak Honsawek
Background: Lumbar spinal canal stenosis is the most common spinal disorder in elderly patients. Canal narrowing is partly the result from hyperthrophy of ligamentum flavum (LF), which mechanically compresses nerve roots. Basic fibroblast growth factor (bFGF) is a potent regulator of many cellular functions including proliferation, differentiation, wound healing, and angiogenesis.
Objective: We examined the pattern of bFGF expression in the ligamentum flavum of lumbar spinal stenosis patients.
Methods: We quantified and localized bFGF expression in LF tissues obtained during surgery from nineteen patients with lumbar spinal stenosis. bFGF expression was determined using quantitative real-time polymerase chain reaction (real-time PCR). The concentration of bFGF was analyzed by enzyme-linked immunosorbent assay.
Results: The bFGF expression was significantly higher in hypertrophic LF of spinal stenosis than that in nonpathologic LF of controls. In real-time PCR, the expression of bFGF was substantially higher in the hypertrophic LF than in controls (P<0.001). The average concentration of bFGF in the hypertrophic LF (mean, 256.7 pg/mg protein) was markedly elevated compared with that of controls (mean, 75.7 pg/mg protein) (P=0.003). There was greater bFGF expression in lumbar spinal stenosis patients as quantified by real-time PCR and enzyme-linked immunosorbent assay.
Conclusion: Our study reveals that increased bFGF expression may be associated with degenerative changes of hypertrophic LF. This suggests that bFGF may contribute to pathogenesis of lumbar spinal stenosis.
Background: Biliary atresia (BA) is a severe neonatal liver disease characterized by progressive fibrosclerotic obliteration of the extrahepatic biliary tree.
Objective: We compared serum adiponectin in post Kasai BA patients with healthy controls and associate adiponectin with clinical outcomes of BA patients.
Methods: One hundred and six postoperative BA patients and 40 controls were recruited in this study. BA patients were categorized into two groups based on their serum total bilirubin (TB) levels (TB<2 mg/dL, no jaundice vs. TB ≥2 mg/dL, persistent jaundice) and alanine aminotransferase (ALT) levels (ALT<45 IU/L, normal ALT vs. ALT ≥45 IU/L, elevated ALT). Serum adiponectin levels were determined by enzyme-linked immunosorbent assay.
Results: BA patients had higher serum adiponectin levels than healthy controls (172.8±90.9 vs. 93.9±53.5 ng/mL, p <0.001). Serum adiponectin levels were elevated in BA patients with jaundice compared to those without jaundice (229.6±89.0 vs. 139.7±74.5 ng/mL, p <0.001). Furthermore, BA patients with elevated ALT displayed significantly higher levels of serum adiponectin than those with normal ALT (187.2±91.8 vs. 117.6±62.8 ng/mL, p <0.001). Additionally, BA patients with portal hypertension had substantially higher serum adiponectin than those without portal hypertension and a poorer clinical outcome (207.0±90.2 vs. 118.5±62.1 ng/mL, p <0.001).
Conclusions: Increased serum adiponectin was associated with a poor outcome in postoperative BA patients. Serum adiponectin might be utilized as a biochemical indicator reflecting the deterioration of liver function and poorer outcome in BA after Kasai operation.
Pobe Luangjarmekorn, Pravit Kitidumrongsuk and Sittisak Honsawek
Free flap surgery is an essential tool in limb reconstruction, but complex and often followed by complications, with many cases requiring additional procedures.
To analyze postoperative complications and need for secondary surgery after free flap surgery over a 10-year period at King Chulalongkorn Memorial Hospital.
We retrospectively reviewed data from a cohort of patients who underwent free flap surgery for limb reconstruction from 2004 to 2014.
We included 35 free flap operations in 29 patients. Mean follow-up time was 6.4 y. Free flap surgical procedures included 7 gracilis transfers, 8 toe transfers, 5 latissimus dorsi flaps, 5 fibular transfers, 4 anterolateral thigh flaps, 2 lateral-arm flaps, 2 radial forearm flaps, and 2 venous free flaps. There were 4 categories of postoperative complications. (1) Patients were those who developed total flap loss after free flap surgery (7/35 flaps, 20%). (2) Patients had major complications requiring additional operations (11/35 flaps, 31%). Major complications included partial flap necrosis, wound swelling with delayed closure, arterial occlusion, postoperative bleeding, infection, and failed implant fixation. (3) Patients had minor complications that required no additional surgical procedures (8/33 flaps, 23%). (4) Patients with no postoperative complications (9/35 flaps, 26%). Secondary surgery after initial free flap was 51% overall (18/35 flaps). The 3 most common secondary procedures included second flap coverage, skin graft, and anastomosis revision. We found free flap surgery performed during the subacute period (14–90 d after injury) to have significantly (P = 0.028) more complications (categories 1 and 2) than surgery performed during the acute period (<14 d) or late reconstruction (>90 d).
Physicians should be prepared for a range of outcomes of free flap surgery and advise their patients of the risk of additional operations accordingly.
Silk fibroin (SF) can be processed into a hydrogel. SF/collagen hydrogel may be a suitable biomaterial for bone tissue engineering.
To investigate in vitro biocompatibility and osteogenic potential of encapsulated rat bone marrow-derived mesenchymal stem cells (rat MSCs) in an injectable Thai SF/collagen hydrogel induced by oleic acid–poloxamer 188 surfactant mixture in an in vitro pilot study.
Rat MSCs were encapsulated in 3 groups of hydrogel scaffolds (SF, SF with 0.05% collagen [SF/0.05C], and SF with 0.1% collagen [SF/0.1C]) and cultured in a growth medium and an osteogenic induction medium. DNA, alkaline phosphatase (ALP) activity, and calcium were assayed at periodically for up to 5 weeks. After 6 weeks of culture the cells were analyzed by scanning electron microscopy and energy dispersive spectroscopy.
Although SF hydrogel with collagen seems to have less efficiency to encapsulate rat MSCs, their plateau phase growth in all hydrogels was comparable. Inability to maintain cell viability as cell populations declined over 1–5 days was observed. Cell numbers then plateaued and were maintained until day 14 of culture. ALP activity and calcium content of rat MSCs in SF/collagen hydrogels were highest at day 21. An enhancing effect of collagen combined with the hydrogel was observed for proliferation and matrix formation; however, benefits of the combination on osteogenic differentiation and biomineralization are as yet unclear.
Rat MSCs in SF and SF/collagen hydrogels showed osteogenic differentiation. Accordingly, these hydrogels may serve as promising scaffolds for bone tissue engineering.
Background: Demineralized bone matrix (DBM) is extensively used in orthopedic, periodontal, and maxillofacial application and investigated as a material to induce new bone formation. Small intestinal submucosa (SIS) derived from the submucosa layer of porcine intestine has widely utilized as biomaterial with minimum immune response. Objectives: Determine the osteoinductive potential of SIS, DBM, SIS/DBM composites in the in vitro cell culture and in vivo animal bioassays for bone tissue engineering. Materials and methods: Human periosteal (HPO) cells were treated in the absence or presence SIS, DBM, and SIS/DBM. Cell proliferation was examined by direct cell counting. Osteoblast differentiation of the HPO cells was analyzed with alkaline phosphatase activity assay. The Wistar rat muscle implant model was used to evaluate the osteoinductive potential of SIS, DBM, and SIS/DBM composites. Results: HPO cells could differentiate along osteogenic lineage when treated with either DBM or SIS/DBM. SIS/ DBM had a tendency to promote more cellular proliferation and osteoblast differentiation than the other treatments. In Wistar rat bioassay, SIS showed no new bone formation and the implants were surrounded by fibrous tissues. DBM demonstrated new bone formation along the edge of old DBM particles. SIS/DBM composite exhibited high osteoinductivity, and the residual SIS/DBM was surrounded by osteoid-like matrix and newly formed bone. Conclusion: DBM and SIS/DBM composites could retain their osteoinductive capability. SIS/DBM scaffolds may provide an alternative approach for bone tissue engineering.