Human periodontal ligament stem cell encapsulation in alginate-fibrin-platelet lysate microbeads for dental and craniofacial regeneration.

OBJECTIVE: Tissue engineering is promising for dental and craniofacial regeneration. The objectives of this study were to develop a novel xeno-free alginate-fibrin-platelet lysate hydrogel with human periodontal ligament stem cells (hPDLSCs) for dental regeneration, and to investigate the proliferation and osteogenic differentiation of hPDLSCs using hPL as a cell culture nutrient supplement. METHODS: hPDLSCs were cultured with Dulbecco's modified eagle medium (DMEM), DMEM + 10% fetal bovine serum (FBS), and DMEM + hPL (1%, 2.5%, and 5%). hPDLSCs were encapsulated in alginate-fibrin microbeads (Alg+Fib), alginate-hPL microbeads (Alg+hPL), or alginate-fibrin-hPL microbeads (Alg+Fib+hPL). hPDLSCs encapsulated in alginate microbeads were induced with an osteogenic medium containing hPL or FBS. Quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) activity, ALP staining, and alizarin red (ARS) staining was investigated. RESULTS: hPDLSCs were released faster from Alg+Fib+hPL than from Alg+hPL. At 14 days, ALP activity was 44.1 ± 7.61 mU/mg for Alg+Fib+hPL group, higher than 28.07 ± 5.15 mU/mg of Alg+Fib (p<0.05) and 0.95 ± 0.2 mU/mg of control (p<0.01). At 7 days, osteogenic genes (ALP, RUNX2, COL1, and OPN) in Alg+Fib+hPL and Alg+Fib were 3-10 folds those of control. At 21 days, the hPDLSC-synthesized bone mineral amount in Alg+Fib+hPL and Alg+Fib was 7.5 folds and 4.3 folds that of control group, respectively. CONCLUSIONS: The 2.5% hPL was determined to be optimal for hPDLSCs. Adding hPL into alginate hydrogel improved the viability of the hPDLSCs encapsulated in the microbeads. The hPL-based medium enhanced the osteogenic differentiation of hPDLSCs in Alg+Fib+hPL construct, showing a promising xeno-free approach for delivering hPDLSCs to enhance dental, craniofacial and orthopedic regenerations.

Novel calcium phosphate cement with biofilm-inhibition and platelet lysate delivery to enhance osteogenesis of encapsulated human periodontal ligament stem cells.

Osteomyelitis is caused by Staphylococcus aureus (S. aureus), with associated progressive bone loss. This study developed for the first time a calcium phosphate cement (CPC) for delivery of doxycycline (DOX) and human platelet lysate (hPL) to fight against S. aureus infection and enhance the osteogenesis of human periodontal ligament stem cells (hPDLSCs). Chitosan-containing CPC scaffolds were fabricated in the absence (CPCC) or presence of DOX (CPCC+DOX). In addition, hPL was encapsulated in alginate microbeads and incorporated into CPCC+DOX (CPCC+DOX+ hPL). Flexural strength of CPCC+DOX + hPL was (5.56 ± 0.55) MPa, lower than (8.26 ± 1.6) MPa of CPCC+DOX (p < 0.05), but exceeding the reported strength of cancellous bone. CPCC+DOX and CPCC+DOX + hPL exhibited strong antibacterial activity against S. aureus, reducing biofilm CFU by 4 orders of magnitude. The hPDLSCs encapsulated in microbeads were co-cultured with the CPCs. The hPDLSCs were able to be released from the microbeads and showed a high proliferation rate, increasing by about 8 folds at 14 days for all groups. The hPL was released from the scaffold and promoted the osteogenic differentiation of hPDLSCs. ALP activity was 28.07 ± 5.15 mU/mg for CPCC+DOX + hPL, higher than 17.36 ± 2.37 mU/mg and 1.34 ± 0.37 mU/mg of CPCC+DOX and CPCC, respectively (p < 0.05). At 7 days, osteogenic genes (ALP, RUNX2, COL-1, and OPN) in CPCC+DOX + hPL were 3-10 folds those of control. The amount of hPDLSC-synthesized bone mineral with CPCC+DOX + hPL was 3.8 folds that of CPCC (p < 0.05). In summary, the novel CPC + DOX + hPL-hPDLSCs scaffold exhibited strong antibacterial activity, excellent cytocompatibility and hPDLSC osteogenic differentiation, showing a promising approach for treatment and prevention of bone infection and enhancement of bone regeneration.

Antibacterial calcium phosphate cement with human periodontal ligament stem cell-microbeads to enhance bone regeneration and combat infection.

Infectious bone defects remain a significant challenge in orthopedics and dentistry. Calcium phosphate cement (CPC) have attracted significant interest in use as local drug delivery system, which with great potential to control release of antibiotics for the treatment of infectious bone defects. Within the current study, a novel antibacterial scaffold of chitosan-reinforced calcium phosphate cement delivering doxycycline hyclate (CPCC + DOX) was developed. Furthermore, the capacity of CPCC + DOX scaffolds for bone regeneration was enhanced by the human periodontal ligament stem cells (hPDLSCs) encapsulated in alginate beads. CPCC + DOX scaffolds were fabricated to contain different concentrations of DOX. Flexural strength of CPCC + DOX ranged from 5.56 ± 0.70 to 6.2 ± 0.72 MPa, which exceeded the reported strength of cancellous bone. Scaffolds exhibited continual DOX release, reaching 80% at 21 days. Scaffold with 5 mg/ml DOX (CPCC + DOX5mg) had a strong antibacterial effect, with a 4-log colony forming unit reduction against S. aureus and P. gingivalis. The proliferation and osteogenic differentiation of hPDLSCs encapsulated in alginate hydrogel microbeads were investigated in culture with CPCC + DOX scaffolds. CPCC + DOX5mg had no negative effect on proliferation of hPDLSCs. Alkaline phosphatase activity, mineral synthesis, and osteogenic gene expressions for CPCC + DOX5mg group were much higher than control group. DOX did not compromise the osteogenic induction. In summary, the novel CPCC + DOX scaffold exhibited excellent mechanical properties and strong antibacterial activity, while supporting the proliferation and osteogenic differentiation of hPDLSCs. The CPCC + DOX + hPDLSCs construct is promising to enhance bone regeneration and combat bone infections in dental, craniofacial, and orthopedic applications.

[Observation of dendrite osteocytes of mice at different developmental stages using Ploton silver staining and phalloidin staining].

OBJECTIVE: To assess the value of Ploton silver staining and phalloidin-iFlour 488 staining in observation of the morphology of osteocyte dendrites of mice at different developmental stages. METHODS: The humerus and femurs were harvested from mice at 0 (P0), 5 (P5), 15 (P15), 21 (P21), 28 (P28), and 35 days (P35) after birth to prepare cryo-sections and paraffin sections. HE staining of P35 mouse femur sections served as a reference for observing osteocytes in the trabecular bone and cortical bone. The humeral sections at different developmental stages were stained with Ploton silver staining to observe the morphology of osteocytes and canaliculi, and the canalicular lengths in the cortical and trabecular bones of the humerus of the mice in each developmental stage were recorded. The cryo-sections of the humerus from P10 and P15 mice were stained with phalloidin iFlour-488 to observe the morphology of osteocytes and measurement of the length of osteocyte dendrites in the cortical bone. RESULTS: In the trabecular bone of the humerus of P0-P15 mice, Ploton silver staining only visualized the outline of the osteocytes, and the morphology of the canaliculi was poorly defined. In P21 or older mice, Ploton silver staining revealed the morphology of the trabecular bone osteocytes and the canaliculi, which were neatly arranged and whose lengths increased significantly with age (P21 vs P28, P < 0.05; P21 vs P35, P < 0.05). In the humeral cortical bone of P15 mice, the morphology of the osteocytes and canalicular could be observed with Ploton silver staining, and the length of the regularly arranged canaliculi of the osteocytes increased significantly with age (P15 vs P21, P < 0.005; P15 vs P28, P < 0.0001; P15 vs P35, P < 0.0001). Phalloidin iFlour-488 staining was capable of visualizing the complete morphology of the osteocytes at P10 and P15; the osteocyte dendrites elongated progressively with age (P10 vs P15, P < 0.01) to form connections with the surrounding osteocytes. CONCLUSIONS: Mouse osteocyte dendrites elongate progressively and their arrangement gradually becomes regular with age. Ploton silver staining can clearly visualize the morphology of the osteocytes and the canaliculi in adult mice but not in mice in early stages of development. Phalloidin iFlour-488 staining for labeling the cytoskeleton can be applied for mouse osteocytes at all developmental stages and allows morphological observation of mouse osteocytes in early developmental stages.

Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet-rich plasma.

Macroporous calcium phosphate cement (CPC) with stem cell seeding is promising for bone regeneration. The objective of this study was to investigate the effects of co-delivering autologous bone marrow mesenchymal stem cells (BMSCs) and autologous platelet-rich plasma (PRP) in CPC scaffold for bone regeneration in minipigs for the first time. Twelve female adult Tibet minipigs (12-18 months old) were used. A cylindrical defect with 10 mm height and 8 mm diameter was prepared at the femoral condyle. Two bone defects were created in each minipig, one at each side of the femoral condyle. Three constructs were tested: (1) CPC scaffold (CPC control); (2) CPC seeded with BMSCs (CPC-BMSC); (3) CPC seeded with BMSCs and PRP (CPC-BMSC-PRP). Two time points were tested: 6 and 12 weeks (n = 4). Good integration of implant with surrounding tissues was observed in all groups. At 12 weeks, the CPC-BMSC-PRP group had significantly less residual CPC remaining in the defect than the CPC-BMSC group and the CPC control (p < 0.05). The residual CPC volume for the CPC-BMSC-PRP group was half that of the CPC control. New bone formation for CPC-BMSC-PRP was more than two-fold that of the CPC control (p < 0.05). CPC-BMSC-PRP had new blood vessel density that was nearly two-fold that of the CPC control (p < 0.05). In conclusion, CPC scaffold with autologous BMSC-PRP doubled the new bone regeneration and blood vessel density in minipigs compared with the CPC control. In the present study, the new macroporous CPC system with co-delivered BMSC-PRP has been shown to promote scaffold resorption and bone regeneration in large defects. Copyright © 2017 John Wiley & Sons, Ltd.

Minipig-BMSCs Combined with a Self-Setting Calcium Phosphate Paste for Bone Tissue Engineering.

Calcium phosphate cements (CPCs) are a new generation of bone repair materials with good biocompatibility for various stem cells. The minipig is a recommended large animal model for bone engineering research. This study aimed to evaluate the feasibility of utilizing CPC scaffolds for the adhesion, proliferation, and osteogenic differentiation of minipig's bone marrow mesenchymal stem cells (pBMSCs). Passage 3 pBMSCs were seeded on the CPC scaffold and cultured with osteogenic culture medium (osteogenic group) or normal medium (control group). The density of viable cells increased in both groups, and pBMSCs firmly attached and spread well on the CPC scaffold. The alkaline phosphatase (ALP) activity in the osteogenic group had significantly increased on day 7 (D7) and peaked on D14. qRT-PCR revealed that mRNA levels of ALP and three osteogenic marker genes were significantly higher on D4, D7, and D14 in the osteogenic group. Alizarin Red S staining showed a significantly higher degree of bone mineralization from D7, D14 to D21 in the osteogenic group. These results indicated that pBMSCs can attach, proliferate well on CPC scaffold, and be successfully induced to differentiate into osteogenic cells. Our findings may be helpful for bone tissue engineering and the studies of bone regeneration.

The effect of extended passaging on the phenotype and osteogenic potential of human umbilical cord mesenchymal stem cells.

Retaining biological characteristics in the extended passaging is crucial for human umbilical cord mesenchymal stem cells (hUCMSCs) in tissue engineering. We aimed to assess morphology, viability, MSC marker expression, and osteogenic activity of hUCSMCs after extended passaging. Passages 4 (P4) and 16 (P16) hUCMSCs displayed similar morphology and viability. The flow cytometry results showed that CD73, CD90, and CD105 were highly expressed at P1-P16. CD166 expression decreased progressively from 90 % at P2 to 61.5 % at P5 (p < 0.05), followed by stable expression through P16. Results from calcium deposition alkaline phosphatase activity and RT-PCR assay showed that both P4 and P16 hUCMSCs differentiated down an osteogenic lineage, with no significant difference in osteogenic capacity (p < 0.05). High-passage UMCSCs maintained stable expression of MSC CD markers as well as stable osteogenic activity. hUCMSCs may thus be suitable for tissue engineering and regenerative medicine applications.