Three-dimensional printed polylactic acid scaffold integrated with BMP-2 laden hydrogel for precise bone regeneration.

BACKGROUND: Critical bone defects remain challenges for clinicians, which cannot heal spontaneously and require medical intervention. Following the development of three-dimensional (3D) printing technology is widely used in bone tissue engineering for its outstanding customizability. The 3D printed scaffolds were usually accompanied with growth factors, such as bone morphometric protein 2 (BMP-2), whose effects have been widely investigated on bone regeneration. We previously fabricated and investigated the effect of a polylactic acid (PLA) cage/Biogel scaffold as a carrier of BMP-2. In this study, we furtherly investigated the effect of another shape of PLA cage/Biogel scaffold as a carrier of BMP-2 in a rat calvaria defect model and an ectopic ossification (EO) model. METHOD: The PLA scaffold was printed with a basic commercial 3D printer, and the PLA scaffold was combined with gelatin and alginate-based Biogel and BMP-2 to induce bone regeneration. The experimental groups were divided into PLA scaffold, PLA scaffold with Biogel, PLA scaffold filled with BMP-2, and PLA scaffold with Biogel and BMP-2 and were tested both in vitro and in vivo. One-way ANOVA with Bonferroni post-hoc analysis was used to determine whether statistically significant difference exists between groups. RESULT: The in vitro results showed the cage/Biogel scaffold released BMP-2 with an initial burst release and followed by a sustained slow-release pattern. The released BMP-2 maintained its osteoinductivity for at least 14 days. The in vivo results showed the cage/Biogel/BMP-2 group had the highest bone regeneration in the rat calvarial defect model and EO model. Especially, the bone regenerated more regularly in the EO model at the implanted sites, which indicated the cage/Biogel had an outstanding ability to control the shape of regenerated bone. CONCLUSION: In conclusion, the 3D printed PLA cage/Biogel scaffold system was proved to be a proper carrier for BMP-2 that induced significant bone regeneration and induced bone formation following the designed shape.

Comparison of demineralized bone matrix and hydroxyapatite as carriers of Escherichia coli recombinant human BMP-2.

BACKGROUND: Autograft has been widely used in various orthopedic and dental surgery for its superior osteogenicity, osteoinductivity and osteoconductivity. But the available volume of the autograft is limited and the efficacy of it is highly affected by the condition of the patients. Therefore, growth factors such as Escherichia coli bone morphogenetic protein-2 (ErhBMP-2) has been widely used in some countries and regions with various carriers that could affect the effects of the growth factors. Demineralized bone matrix (DBM) has been widely used as a bone graft substitute and growth factor carrier, but its effect as a carrier of ErhBMP-2 was less investigated. MATERIALS AND METHODS: Rat calvaria defect model was used in this study. We implanted ErhBMP-2 with DBM or hydroxyapatite (HA) as a carrier in 8 mm calvaria defect and compared their bone regeneration effect in 4th week and 8th week after implantation with micro-CT and histology. The data was analyzed with one-way ANOVA method with Bonferroni post-hoc analysis. RESULT: The group with DBM as the carrier showed significantly higher bone volume and bone thickness than the groups with HA as the carrier in both weeks. And the histology sections showed less adipose tissue formed in the groups with DBM as the carrier. CONCLUSION: DBM could be a better carrier for ErhBMP-2 than HA.

Osteogenic Response of MC3T3-E1 and Raw264.7 in the 3D-Encapsulated Co-Culture Environment.

BACKGROUND: Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix® 3D culture system, which provides a physiologically relevant environment. METHODS: The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. RESULTS: Treatment with osteoblastic induction factors (+3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D co-culture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK-RANKL signaling, which is important in osteoblast regulation of osteoclasts. CONCLUSION: In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

The toxicological effect of 4-week repeated intravenous injection of activin a/BMP-2 chimera and 2-week recovery study in Beagle dog.

The purposes of this study was to determine the toxicological effect of repeated intravenous administration of Activin A/BMP-2 chimera (AB204) in beagle dogs for a long period of four weeks and evaluate two-week recovery. AB204 was administered at doses of 0.08, 0.16, or 0.32 mg/kg/day to three male and three female beagle dogs for 4 weeks as the experimental group. For the control group, sterile saline was administered to three male and three female beagle dogs. For the two-week recovery test, two male and two female beagle dogs were randomly selected from the control group and the 0.32 mg/kg/day administered experimental group. General symptoms, body weight, food consumption, ophthalmological examination, electrocardiogram, urinalysis, hematology and blood biochemistry, organ weights, autopsy, and histopathological examination were observed or conducted. No animals died. There was no significant difference in any parameter evaluated between the experimental group and the control group. Histopathological examination revealed compound inflammation at the administration site in both the experimental group and the control group. The inflammation disappeared during the two-week recovery. These results indicated that repetitive intravenous injection of AB204 in beagle dog for a long period of four weeks did not show any toxicity. Therefore, no observed adverse effects level (NOAEL) of AB204 was 0.32 mg/kg/day in big animal model.

Umbilical Cord Mesenchymal Stem Cell-Derived Nanovesicles Potentiate the Bone-Formation Efficacy of Bone Morphogenetic Protein 2.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is one of the most potent osteogenic factors used to treat bone loss. However, at higher doses, rhBMP-2 does not necessarily increase bone formation but rather increases the incidence of adverse side effects. Here, we investigated whether umbilical cord mesenchymal stem cell (UCMSC)-derived nanovesicles (NVs) further increase the in vivo bone formation at high doses of rhBMP-2. In the presence of UCMSC-derived NVs, proliferation, migration, and tube formation of human umbilical vein endothelial cells were stimulated in vitro. Furthermore, migration and osteogenesis of human bone marrow-derived mesenchymal stem cells were stimulated. To examine the efficacy of UCMSC-derived NVs on in vivo bone formation, collagen sponges soaked with rhBMP-2 and UCMSC-derived NVs were used in athymic nude mice with calvarial defects. At a high rhBMP-2 dosage (500 ng/mL), UCMSC-derived NVs significantly promoted bone formation in calvarial defects; however, the UCMSC-derived NVs alone did not induce in vivo bone formation. Our results indicate that UCMSC-derived NVs can potentiate the bone formation efficacy of rhBMP-2 at a high dosage.

A novel 3D indirect co-culture system based on a collagen hydrogel scaffold for enhancing the osteogenesis of stem cells.

In this study, the paracrine effect between adipose-derived mesenchymal stem cells (ADSCs) and osteoblasts was investigated in collagen-based three-dimensional (3D) scaffolds. 3D encapsulation of mesenchymal stem cells in hydrogel scaffolds was conducted for bone tissue regeneration. Osteoblasts were encapsulated in alginate microbeads with uniform size, which could be controlled by varying the supply voltage using electrostatic droplet extrusion. Osteoblast-encapsulated microbeads were embedded with ADSCs in collagen bulk hydrogel scaffolds with a high survival rate. The separated space between the two types of cells made it possible to confirm ADSC differentiation into osteogenic lineages in the 3D collagen hydrogel scaffold by the paracrine effect in vitro. Furthermore, co-cultured ADSC and osteoblasts showed enhanced bone formation compared with the ADSC monoculture group in the rat calvarial defect model. The system developed in this study provides a novel in vitro tissue model for bone regeneration without exogenous factors, and it has the potential to be used to study the paracrine effect in various co-culture systems in the near future.

BMP-2 and hMSC dual delivery onto 3D printed PLA-Biogel scaffold for critical-size bone defect regeneration in rabbit tibia.

3D printing technology has various advantages, and the incorporation of bioactive substances into the 3D printed scaffold provides the biological and architectural characteristics of the scaffolds, which is very important for obtaining a good osseointegration effect. In this relation, this study prepared a novel porous hollow cage poly(lactic acid) (PLA) 3D printed scaffold and combined recombinant human bone morphogenetic protein-2 (rhBMP-2) and/or mesenchymal stem cells (MSCs) with Biogel composed of gelatin and alginate. Then, the scaffolds were used to evaluate the resulting bone regeneration through both in vitro and in vivo tests. The experimental group was divided into four groups as follows: only PLA scaffold (PLA); PLA scaffold filled with BMP-2 loaded on Biogel (P-BG-B2); PLA scaffold filled with MSCs encapsulated Biogel (P-BG-M); PLA scaffold filled with both BMP-2 and MSCs loaded on Biogel (P-BG-B2-M). Then in vitro results showed that the PLA-Biogel-based scaffold increased cell proliferation, and the P-BG-B2-M group showed a higher alkaline phosphatase activity and bone-related gene expression than was seen with the P-BG-M group at all the time points. It was shown that four weeks post-operative micro-CT analysis showed that within the defect site the P-BG-B2 group had a significantly higher percent bone volume (BV/TV) than the PLA group and P-BG-M group. And, out of the defect site, the P-BG-B2-M group BV/TV was shown significantly higher than the PLA group (p < 0.05). Histologically, defects in the P-BG-B2-M group showed a homogeneous new bone distribution, however the P-BG-B2 group and P-BG-M group presented a notably higher bone formation in the internal region than in the proximal region of the bone defect site. In conclusion, the 3D PLA-Biogel-based scaffold adapted rhBMP-2 and MSCs with carrier PLA showed good biocompatibility and high possibility as an effective and satisfactory bone graft material.

Enhanced healing of rat calvarial defects with 3D printed calcium-deficient hydroxyapatite/collagen/bone morphogenetic protein 2 scaffolds.

In this paper, we mainly to evaluate the newly formed bone using the Calcium deficient hydroxyapatite (CDHA)/collagen-based bio-ceramic scaffold as Bone Morphogenetic Protein-2 (BMP-2) carrier in rat calvarial critical-sized bone defect. In the real-time PCR analysis, the CDHA/collagen scaffold loaded rhBMP-2 group showed significantly enhanced results of bone-related gene expression (p < 0.05). In the in vivo study, the micro-CT showed that the main bone formation parameters of percent bone volume and trabecular number of the two experiment groups (CDHA/Collagen (CDHA) group, BV/TV: 14.21 ± 3.20, Tb.N: 2.37 ± 0.50; CDHA/Collagen/rhBMP-2(BMP) group, BV/TV: 14.51 ± 3.12, Tb.N: 2.75 ± 0.65) were significantly higher than those of the control (Blank, BV/TV: 3.25 ± 1.25, Tb.N: 0.57 ± 0.20) group (p < 0.05). Although there was no significant difference between the two experimental groups, the BMP group results were slightly higher than those of the CDHA group (p > 0.05). Moreover, the histological results also supported the micro-CT results. The scaffold of CDHA/collagen seems to be a suitable bio-ceramic carrier loaded rhBMP-2, and appears to enhance new bone formation and bone regeneration in bone defect after implantation.

Preparation of beta-tricalcium phosphate microsphere-hyaluronic acid-based powder gel composite as a carrier for rhBMP-2 injection and evaluation using long bone segmental defect model.

The specific objective of this study was to evaluate whether rhBMP-2-loaded bio-scaffolds can be used as effective rhBMP-2 carriers in the implantation of bone defect sites or poor bone quality in host bone. The rhBMP-2 release pattern test showed slow results in both groups, and a 1:9 ratio composition with a high water-absorption rate was selected for in vivo study. All animals euthanized after 9 weeks. The new bone formation and bone quantity and quality of fibular samples were examined. The results showed that the rhBMP-2 powder gel composite improved the new bone formation in the cortical bone and the marrow space. The length of new bone formation ratio of the rhBMP-2 loaded composite group was significantly higher than the powder gel group. The composite of powder gel seems to be a nice carrier, and slow release of rhBMP-2 can promote new bone formation in a segmental cortical bone defect after implantation.