Stem cells combined with three-dimensional scaffolds for the treatment of osteoporotic bone defects北大核心

BACKGROUND: Stem cells combined with a three-dimensional scaffold have great potential for the treatment of osteoporotic bone defects. OBJECTIVE: To introduce the application of stem cells combined with the three-dimensional scaffold in repairing osteoporotic bone defects. METHODS: A computer-based search of PubMed, Web of Science, Springerlink, Medline, WanFang and CNKI databases was performed for relevant articles published from 2007 to 2017 with “stem cells, scaffold, osteoporosis, bone defects” as key words in English and Chinese, repsectively. Initially, 142 articles were retrieved, and finally 45 articles were included in result analysis. RESULTS AND CONCLUSION: Due to the potential of self-renewal and multilineage differentiation, stem cells can be used to repair or regenerate damaged tissues, which are considered as an ideal cell source for the treatment of diseases in orthopedics. The suitable scaffold can provide a favorable microenvironment for repairing the attachment and growth of the cells related to the bone defect, which can promote the healing without additional side effects. Furthermore, stem cells combined with three-dimensional scaffolds provide a promising clinical application for the treatment of osteoporotic bone defects by regulation of bone metabolism. In addition, gene-modified stem cells with three-dimensional scaffolds bring a huge potential in the treatment of osteoporotic bone defects. In conclusion, the combination of stem cells and three-dimensional scaffolds provides a new approach for the treatment of osteoporotic bone defects, which may be one of the future therapeutic strategies. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Combination of BMP-2-releasing gelatin/β-TCP sponges with autologous bone marrow for bone regeneration of X-ray-irradiated rabbit ulnar defects

The objective of this study is to evaluate the feasibility of gelatin sponges incorporating β-tricalcium phosphate (β-TCP) granules (gelatin/β-TCP sponges) to enhance bone regeneration at a segmental ulnar defect of rabbits with X-ray irradiation. After X-ray irradiation of the ulnar bone, segmental critical-sized defects of 20-mm length were created, and bone morphogenetic protein-2 (BMP-2)-releasing gelatin/β-TCP sponges with or without autologous bone marrow were applied to the defects to evaluate bone regeneration. Both gelatin/β-TCP sponges containing autologous bone marrow and BMP-2-releasing sponges enhanced bone regeneration at the ulna defect to a significantly greater extent than the empty sponges (control). However, in the X-ray-irradiated bone, the bone regeneration either by autologous bone marrow or BMP-2 was inhibited. When combined with autologous bone marrow, the BMP-2 exhibited significantly high osteoinductivity, irrespective of the X-ray irradiation. The bone mineral content at the ulna defect was similar to that of the intact bone. It is concluded that the combination of bone marrow with the BMP-2-releasing gelatin/β-TCP sponge is a promising technique to induce bone regeneration at segmental bone defects after X-ray irradiation.     

Hydroxyapatite/beta-tricalcium phosphate scaffolds combined with adipose-derived stem cells for the treatment of spinal defects in rabbits北大核心

BACKGROUND: Repair of bone defects is not only a clinical problem, but also a hot topic in the field of orthopedics. Although autologous bone grafting is considered as the “gold standard” for bone repair, its use is limited due to the limited source of autogenous bone, bone infections and pains that are easy to occur in the donor region. Allograft bones are always associated with immune rejection, slow healing, and infection. Therefore, it is imminent to develop new materials for bone repair. OBJECTIVE: To explore the effect of rabbit adipose-derived stem cells (rADSCs) as seed cells and hydroxyapatite/p-tricalcium phosphate (HA/β-TCP) composite as a carrier on the repair of rabbit vertebral defects. METHODS: Thirty-eight 3-month-old New Zealand white rabbits were selected, ond two of them were used to culture rADSCs in vitro. Passage 3 rADSCc were inoculated on HA/β-TCP scaffolds and then cultured in vitro for 2 weeks. A 5 mmx5 mmx3 mm bone defect was prepared at the anterior edge of L4/5 vertebral body in the remaining 36 rabbits. These model rabbits were then randomized into cell-scaffold composite group, scaffold group and control group with no intervention, with 12 rabbits in each group. rADSCs/HA/β-TCP composite and HA/β-TCP scaffold were implanted into the cell-scaffold and HA/β-TCP groups, respectively. Anteroposterior and lateral DR of the spine and Lane-Sandhu X-ray wera performed at 4, 8, 12 postoperative weeks. All rabbits wera sacrificed at 12 postoperative weeks and specimens were collected for grass and histopathological observations. RESULTS AND CONCLUSION: Under the grass observation, bone defects in the cell-scaffold group were essentially replaced by new bone tissues, which was significantly better than that in the scaffold group and control group. At 12 postoperative weeks, the material implanted was basically absorbed in the cell-scaffold group, partially absorbed in the scaffold group and poorly absorbed in the control group in which there was a clear boundary with the surrounding tissues and patchy calcifed shadows were visible. X-ray results showed that the repair effect in the cell-scaffold group was better than that in the scaffold group and control group (P < 0.05). Histopathological findings showed the marked absorption of the iplant in the cell-scaffold group, partial residual in the scaffold group with some fibrous calluses and osteoid tissues, and a large amount of fiber tissues and a small amount of calluses in the control group. Overall, the rADSCs/HA/β-TCA has a good ability to repair bone defects. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Controllable dual-release of dexamethasone and bovine serum albumin from PLGA/β-tricalcium phosphate composite scaffolds

Localized dual-drug delivery from biodegradable scaffolds is an important strategy in tissue engineering. In this study, porous poly(L-lactide-co-glycolide) (PLGA)/β-tricalcium phosphate scaffolds containing both dexamethasone (Dex) and bovine serum albumin (BSA) were prepared by incorporating Dex-loaded and BSA-loaded microspheres into the scaffolds. PLGA microspheres containing Dex or BSA were prepared by spray-drying and double emulsion/solvent evaporation, respectively. In vitro release studies indicated that microspheres prepared from PLGA in 3:1 molar ratio of L-lactide/glycolide and 89.5 kDa relative molecular mass showed prolonged release profiles compared with those prepared from PLGA in 1:1 L-lactide/glycolide molar ratio and 30.5 kDa relative molecular mass. Additionally, introduction of poly(ethylene glycol) in the PLGA chain could improve the encapsulation efficiency and reduce the release rate. Based on the above results, controllable dual-release of Dex and BSA with relatively higher or lower release rate was achieved by incorporating Dex-loaded and BSA-loaded microspheres with different release profiles into the PLGA/β-tricalcium phosphate scaffolds.     

Fabrication and characterization of novel nano hydroxyapatite/β-tricalcium phosphate scaffolds in three different composition ratios

The biphasic calcium phosphate (BCP) concept was introduced to overcome disadvantages of single phase biomaterials. Different composition ratios of BCP bioceramics have been studied, yet controversies regarding the effects of ratio on biomaterial behavior still exist. In this study, BCP scaffolds were prepared from nano hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) that were synthesized via a solid state reaction. Three different composition ratios of pure BCP and collagen-based BCP scaffolds (%HA/%β-TCP; 30/70, 40/60, and 50/50) were produced using a polymeric sponge method. Physical and mechanical properties of all materials and scaffolds were investigated. SEM showed overall distribution of both macropores (80-200 μm) and micropores (0.5-2 μm) with high interconnected porosities. Total porosity of pure BCP (90% ± 3%) was found to be higher than collagen-based BCP (85% ± 2%). It was observed that following sintering process, dimensional shrinkage of large scaffolds (39% ± 4%) was lower than small ones (42% ± 5%) and scaffolds with high HA ratios (50%) experienced higher dimensional changes than those with higher β-TCP (70%) ratios (45% ± 3% and 36% ± 1%, respectively). Compressive strength of both groups was less than 0.1 MPa and collagen coating had almost no influence on mechanical behavior. Further studies may improve the physical properties of these scaffolds and investigate their exact biological behaviors.     

Osteoinduction of hydroxyapatite/β-tricalcium phosphate bioceramics in mice with a fractured fibula

Many studies have shown that calcium phosphate ceramics can induce bone formation in non-osseous sites without the application of any osteoinductive biomolecules, but the mechanisms of this phenomenon (intrinsic osteoinduction of bioceramics) remain unclear. In this study, we compared the intrinsic osteoinduction of porous hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) implanted in mice at different sites. In 30 mice the left fibula was fractured and the right fibula was kept intact. A porous HA/β-TCP cylinder was implanted into both the left (group 1) and right (group 2) leg muscles of each animal. In addition, two HA/β-TCP cylinders were bilaterally implanted into leg subcutaneous pockets (group 3) in each of the remaining 15 mice. New bone formation was studied in the three groups by histology, histomorphometry and immunostaining. In group 1 new bone was observed at week 6 and bone marrow appeared at week 12. In group 2 new bone was observed at week 8 and bone marrow appeared at week 12. The new bone area percentage in group 1 was significantly higher than in group 2 at both weeks 8 and 12. In contrast, group 3 did not show any new bone within the period studied. These differences were explained based on the location of the implants and thus their proximity to the osteogenic environment of fracture healing. The results support the hypothesis that intrinsic osteoinduction by calcium phosphate ceramics is the result of adsorption of osteoinductive substances on the surface.     

Combinatorial screening of osteoblast response to 3D calcium phosphate/poly(ε-caprolactone) scaffolds using gradients and arrays

There is a need for combinatorial and high-throughput methods for screening cell-biomaterial interactions to maximize tissue generation in scaffolds. Current methods employ a flat two-dimensional (2D) format even though three-dimensional (3D) scaffolds are more representative of the tissue environment in vivo and cells are responsive to topographical differences of 2D substrates and 3D scaffolds. Thus, combinatorial libraries of 3D porous scaffolds were developed and used to screen the effect of nano-amorphous calcium phosphate (nACP) particles on osteoblast response. Increasing nACP content in poly(ε-caprolactone) (PCL) scaffolds promoted osteoblast adhesion and proliferation. The nACP-containing scaffolds released calcium and phosphate ions which are known to activate osteoblast function. Scaffold libraries were fabricated in two formats, gradients and arrays, and the magnitude of the effect of nACP on osteoblast proliferation was greater for arrays than gradients. The enhanced response in arrays can be explained by differences in cell culture designs, diffusional effects and differences in the ratio of "scaffold mass to culture medium". These results introduce a gradient library approach for screening large pore 3D scaffolds and demonstrate that inclusion of the nACP particles enhances osteoblast proliferation in 3D scaffolds. Further, comparison of gradients and arrays suggests that gradients were more sensitive for detecting effects of scaffold composition on cell adhesion (short time points, 1 day) whereas arrays were more sensitive at detecting effects on cell proliferation (longer time points, 14 day).     

Balancing mechanical strength with bioactivity in chitosan–calcium phosphate 3D microsphere scaffolds for bone tissue engineering: air- vs. freeze-drying processes

The objective of this study was to evaluate the potential benefit of 3D composite scaffolds composed of chitosan and calcium phosphate for bone tissue engineering. Additionally, incorporation of mechanically weak lyophilized microspheres within those air-dried (AD) was considered for enhanced bioactivity. AD microsphere, alone, and air- and freeze-dried microsphere (FDAD) 3D scaffolds were evaluated in vitro using a 28-day osteogenic culture model with the Saos-2 cell line. Mechanical testing, quantitative microscopy, and lysozyme-driven enzymatic degradation of the scaffolds were also studied. FDAD scaffold showed a higher concentration (p?<?0.01) in cells per scaffold mass vs. AD constructs. Collagen was ～31% greater (p?<?0.01) on FDAD compared to AD scaffolds not evident in microscopy of microsphere surfaces. Alternatively, AD scaffolds demonstrated a superior threefold increase in compressive strength over FDAD (12 vs. 4?MPa) with minimal degradation. Inclusion of FD spheres within the FDAD scaffolds allowed increased cellular activity through improved seeding, proliferation, and extracellular matrix production (as collagen), although mechanical strength was sacrificed through introduction of the less stiff, porous FD spheres.     

In vitro 3D culture of human chondrocytes using modified ε-caprolactone scaffolds with varying hydrophilicity and porosity

Two series of 3D scaffolds based on ε-caprolactone were synthesized. The pore size and architecture (spherical interconnected pores) was the same in all the scaffolds. In one of the series of scaffolds, made of pure ε-polycaprolactone, the volume fraction of pores varied between 60% and 85% with the main consequence of varying the interconnectivity between pores since the pore size was kept constant. The other scaffolds were prepared with copolymers made of a ε-caprolactone-based hydrophobous monomer and hydroxyethyl acrylate, as the hydrophilic component. Thus, the hydrophilicity and, presumably, the adhesion properties varied monotonously in the copolymer series while porosity was kept constant. A suspension of human chondrocytes in culture medium was injected in the 3D scaffolds and cultured in static conditions up to 28 days. SEM and immunofluorescence assays allowed characterizing cells and extracellular matrix inside the scaffolds after different culture times. To do that, cross sections of the scaffolds were observed by SEM and confocal microscopy. The quantity of cells inside the scaffolds decreases with a decrease of the volume fraction of pores, due to the lack of interconnectivity between the cavities. The scaffolds up to a 30% of hydrophilicity behave in a similar way than the hydrophobous; a further increase of the hydrophilicity rapidly decreases cell viability. In all the experiments production of collagen type I, type II, and aggrecan was found, and some cells were Ki-67 positive, showing that some cells are adhered to the pore walls and maintain their dedifferentiated phenotype even when cultured in three-dimensional conditions.     

The enhancement of bone regeneration by a combination of osteoconductivity and osteostimulation using β-CaSiO3/β-Ca3(PO4)2 composite bioceramics

β-Tricalcium phosphate (β-TCP) is osteoconductive, while β-calcium silicate (β-CS) is bioactive with osteostimulative properties. Porous β-CaSiO₃/β-Ca₃(PO₄)₂ composite bioceramic scaffolds with various β-TCP:β-CS ratios were designed to combine both osteoconductivity and osteostimulation in order to enhance bone regeneration. The composite scaffolds were implanted in critical sized femur defects (6 × 12 mm) for 4, 12 and 26 weeks with pure β-TCP and β-CS scaffolds as the controls. The in vivo biodegradation and bone regeneration of the specimens were investigated using sequential histological evaluations, immunohistochemical examination and micro-computed tomography technology. The results showed that the scaffolds with 50 and 80 wt.% β-CS dramatically enhanced the amount of newly formed bone and reduced the degradation rate. In contrast, porous β-CS displayed poor new bone formation due to its rapid degradation, while porous β-TCP showed moderate bone regeneration starting on the surface of the implants, due to a lack of osteostimulation. More importantly, the scaffolds with 50 and 80 wt.% β-CS not only had excellent osteoconductivity, but also stimulated rapid bone formation, and they could degrade progressively at a rate matching the regeneration of new bone. In summary, our findings indicated that the degradation rate and bioactivity of β-CS/β-TCP composite bioceramic scaffolds could be adjusted by controlling the ratio of β-CS to β-TCP, suggesting the potential application of β-CS/β-TCP composite bioceramic scaffolds with 50 and 80 wt.% β-CS component in hard tissue regeneration and bone tissue engineering.     

Value of cytokeratin 5/6 immunostaining using D5/16 B4 antibody in the spectrum of proliferative intraepithelial lesions of the breast. A comparative study with 34βE12 antibody

Previous studies have shown that basal-type cytokeratins (CKs) can distinguish usual ductal hyperplasia (UDH) from the spectrum of atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). Indeed, expression of these CKs is weak or absent in ADH, DCIS and LCIS. However, the diagnostic usefulness of D5/16B4 antibody (anti-CK5/6) has never been compared with that of 34betaE12 antibody (anti-CK1/5/10/14). We performed immunostaining of CK 5/6 and CK1/5/10/14 on 100 breast lesions, including UDH ( n=31), ADH ( n=5), DCIS ( n=54) and LCIS ( n=10). Abundant immunostaining was observed in all UDH using both antibodies. Four of five of the ADH cases showed less than 5% of CK5/6 stained cells, the remaining case showed 30% of labeled cells. With 34betaE12 antibody, three of five of the ADH cases showed less than 5% labeled cells, while two cases showed more than 30% of stained cells. None of the 54 DCIS or the 10 LCIS was labeled by D5/16B4, while a lack of 34betaE12 immunostaining was observed in only 15 of 54 DCIS and 2 of 10 LCIS. We confirmed that D5/16B4 antibody directed against CK5/6 is useful in distinguishing UDH from the spectrum of ADH/DCIS/LCIS. We also demonstrated that D5/16B4 is far a more specific marker than 34betaE12 antibody.