A low dose of 1μg rhBMP-2 was immobilised by four different functionalising techniques on recently developed poly(l-lactide)-co-(ε-caprolactone) [(poly(LLA-co-CL)] scaffolds. It was either (i) physisorbed on unmodified scaffolds [PHY], (ii) physisorbed onto scaffolds modified with nanodiamond particles [nDP-PHY], (iii) covalently linked onto nDPs that were used to modify the scaffolds [nDP-COV] or (iv) encapsulated in microspheres distributed on the scaffolds [MICS]. Release kinetics of BMP-2 from the different scaffolds was quantified using targeted mass spectrometry for up to 70days. PHY scaffolds had an initial burst of release while MICS showed a gradual and sustained increase in release. In contrast, NDP-PHY and nDP-COV scaffolds showed no significant release, although nDP-PHY scaffolds maintained bioactivity of BMP-2. Human mesenchymal stem cells cultured in vitro showed upregulated BMP-2 and osteocalcin gene expression at both week 1 and week 3 in the MICS and nDP-PHY scaffold groups. These groups also demonstrated the highest BMP-2 extracellular protein levels as assessed by ELISA, and mineralization confirmed by Alizarin red. Cells grown on the PHY scaffolds in vitro expressed collagen type 1 alpha 2 early but the scaffold could not sustain rhBMP-2 release to express mineralization. After 4weeks post-implantation using a rat mandible critical-sized defect model, micro-CT and Masson trichrome results showed accelerated bone regeneration in the PHY, nDP-PHY and MICS groups. The results demonstrate that PHY scaffolds may not be desirable for clinical use, since similar osteogenic potential was not seen under both in vitro and in vivo conditions, in contrast to nDP-PHY and MICS groups, where continuous low doses of BMP-2 induced satisfactory bone regeneration in both conditions. The nDP-PHY scaffolds used here in critical-sized bone defects for the first time appear to have promise compared to growth factors adsorbed onto a polymer alone and the short distance effect prevents adverse systemic side effects.
IntroductionA major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene-expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation.MethodsMSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining.ResultsThe results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks.ConclusionsThis study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs.
Significant evidence has indicated that poly(L-lactide)-co-(ɛ-caprolactone) [(poly(LLA-co-CL)] scaffolds could be one of the suitable candidates for bone tissue engineering. Oxygen-terminated nanodiamond particles (n-DP) were combined with poly(LLA-co-CL) and revealed to be positive for cell growth. In this study, we evaluated the influence of poly(LLA-co-CL) scaffolds modified by n-DP on attachment, proliferation, differentiation of bone marrow stromal cells (BMSCs) in vitro, and on bone formation using a sheep calvarial defect model. BMSCs were seeded on either poly(LLA-co-CL)- or n-DP-coated scaffolds and incubated for 1 h. Scanning electron microscopy (SEM) and fluorescence microscopy were used in addition to protein and DNA measurements to evaluate cellular attachment on the scaffolds. To determine the effect of n-DP on proliferation of BMSCs, cell/scaffold constructs were harvested after 3 days and evaluated by Bicinchoninic Acid (BCA) protein assay and SEM. In addition, the osteogenic differentiation of cells grown for 2 weeks on the various scaffolds and in a dynamic culture condition was evaluated by real-time RT-PCR. Unmodified and modified scaffolds were implanted into the calvaria of six-year-old sheep. The expression of collagen type I (COL I) and bone morphogenetic protein-2 (BMP-2) after 4 weeks as well as the formation of new bone after 12 and 24 weeks were analyzed by immunohistochemistry and histology. Scaffolds modified with n-DP supported increased cell attachment and the mRNA expression of osteopontin (OPN), bone sialoprotein (BSP), and BMP-2 were significantly increased after 2 weeks of culture. The BMSCs had spread well on the various scaffolds investigated after 3 days in the study with no significant difference in cell proliferation. Furthermore, the in vivo data revealed more positive staining of COL I and BMP-2 in relation to the n-DP-coated scaffolds after 4 weeks and presented more bone formation after 12 and 24 weeks. n-DP modification significantly increased cell attachment and differentiation of BMSCs on poly(LLA-co-CL) scaffolds in vitro and enhanced bone formation in vivo.
Constructs intended for bone tissue engineering (TE) are influenced by the initial cell seeding density. Therefore, the objective of this study was to determine the effect of bone marrow stromal stem cells (BMSCs) density loaded onto copolymer scaffolds on bone regeneration. BMSCs were harvested from rat's bone marrow and cultured in media with or without osteogenic supplements. Cells were seeded onto poly(l‐lactide‐co‐ε‐caprolactone) [poly(LLA‐co‐CL)] scaffolds at two different densities: low density (1 × 106 cells/scaffold) or high density (2 × 106 cells/scaffold) using spinner modified flasks and examined after 1 and 3 weeks. Initial attachment and spread of BMSC onto the scaffolds was recorded by scanning electron microscopy. Cell proliferation was assessed by DNA quantification and cell differentiation by quantitative real‐time reverse transcriptase‐polymerized chain reaction analysis (qRT‐PCR). Five‐millimeter rat calvarial defects (24 defects in 12 rats) were implanted with scaffolds seeded with either low or high density expanded with or without osteogenic supplements. Osteogenic supplements significantly increased cell proliferation (p < 0.001). Scaffolds seeded at high cell density exhibited higher mRNA expressions of Runx2 p = 0.001, Col1 p = 0.001, BMP2 p < 0.001, BSP p < 0.001, and OC p = 0.013. More bone was formed in response to high cell seeding density (p = 0.023) and high seeding density with osteogenic medium (p = 0.038). Poly (LLA‐co‐CL) scaffolds could be appropriate candidates for bone TE. The optimal number of cells to be loaded onto scaffolds is critical for promoting Extracellular matrix synthesis and bone formation. Cell seeding density and osteogenic supplements may have a synergistic effect on the induction of new bone. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3649–3658, 2015.
IntroductionRapid establishment of functional blood vessels is a prerequisite for successful tissue engineering. During vascular development, endothelial cells (ECs) and perivascular cells assemble into a complex regulating proliferation of ECs, vessel diameter and production of extracellular matrix proteins. The aim of this study was to evaluate the ability of mesenchymal stem cells (MSCs) to establish an endothelial-perivascular complex in tissue-engineered constructs comprising ECs and MSCs.MethodsPrimary human ECs and MSCs were seeded onto poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) scaffolds and grown in dynamic culture before subcutaneous implantation in immunocompromised mice for 1 and 3 weeks. Cellular activity, angiogenic stimulation and vascular assembly in cell/scaffold constructs seeded with ECs or ECs/MSCs in a 5:1 ratio was monitored with real-time RT-PCR, ELISA and immunohistochemical microscopy analysis.ResultsA quiescent phenotype of ECs was generated, by adding MSCs to the culture system. Decreased proliferation of ECs, in addition to up-regulation of selected markers for vascular maturation was demonstrated. Baseline expression of VEGFa was higher for MSCs compared with EC (P <0.001), with subsequent up-regulated VEGFa-expression for EC/MSC constructs before (P <0.05) and after implantation (P <0.01). Furthermore, an inflammatory response with CD11b + cells was generated from implantation of human cells. At the end of the 3 week experimental period, a higher vascular density was shown for both cellular constructs compared with empty control scaffolds (P <0.01), with the highest density of capillaries being generated in constructs comprising both ECs and MSCs.ConclusionsInduction of a quiescent phenotype of ECs associated with vascular maturation can be achieved by co-seeding with MSCs. Hence, MSCs can be appropriate perivascular cells for tissue-engineered constructs.
The aim is to evaluate the effect of modifying poly[( L -lactide)-co-(ε-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modifi ed scaffolds degrade faster than the unmodifi ed. Gene analysis at week 1 shows highest expression of proinfl ammatory markers around nDP scaffolds; although the presence of infl ammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fi brous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1α2, and ANGPT1 are signifi cantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates infl ammation while lowering the dose of BMP-2 to a relatively safe and economical level.
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