Thilo Waag

Release and bioactivity of bone morphogenetic protein-2 are affected by scaffold binding techniques in vitro and in vivo

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.

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity.

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure-activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei.

Biofunctionalization of scaffold material with nano-scaled diamond particles physisorbed with angiogenic factors enhances vessel growth after implantation

UNLABELLED Biofunctionalized scaffold facilitates complete healing of large defects. Biological constraints are induction and ingrowth of vessels. Angiogenic growth factors such as vascular endothelial growth factor or angiopoietin-1 can be bound to nano-scaled diamond particles. Corresponding bioactivities need to be examined after biofunctionalization. We therefore determined the physisorptive capacity of distinctly manufactured, differently sized nDP and the corresponding activities of bound factors. The properties of biofunctionalized nDPs were investigated on cultivated human mesenchymal stem cells and on the developing chicken embryo chorio-allantoic membrane. Eventually porous bone substitution material was coated with nDP to generate an interface that allows biofactor physisorption. Angiopoietin-1 was applied shortly before scaffold implantation into an osseous defect in sheep calvaria. Biofunctionalized scaffolds exhibited significantly increased rates of angiogenesis already one month after implantation. Conclusively, nDP can be used to ease functionalization of synthetic biomaterials. FROM THE CLINICAL EDITOR With the advances in nanotechnology, many nano-sized materials have been used in the biomedical field. This is also true for nano-diamond particles (nDP). In this article, the authors investigated the physical properties of functionalized nano-diamond particles in both in-vitro and in-vivo settings. The positive findings would help improve understanding of these nanomaterials in regenerative medicine.

In Vivo Host Response and Degradation of Copolymer Scaffolds Functionalized with Nanodiamonds and Bone Morphogenetic Protein 2.

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 modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory 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 fibrous 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 significantly 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 inflammation while lowering the dose of BMP-2 to a relatively safe and economical level.

A Copolymer Scaffold Functionalized with Nanodiamond Particles Enhances Osteogenic Metabolic Activity and Bone Regeneration.

Functionalizing polymer scaffolds with nanodiamond particles (nDPs) has pronounced effect on the surface properties, such as improved wettability, an increased active area and binding sites for cellular attachment and adhesion, and increased ability to immobilize biomolecules by physical adsorption. This study aims to evaluate the effect of poly(l-lactide-co-ε-caprolactone) (poly(LLA-co-CL)) scaffolds, functionalized with nDPs, on bone regeneration in a rat calvarial critical size defect. Poly(LLA-co-CL) scaffolds functionalized with nDPs are also compared with pristine scaffolds with reference to albumin adsorption and seeding efficiency of bone marrow stromal cells (BMSCs). Compared with pristine scaffolds, the experimental scaffolds exhibit a reduction in albumin adsorption and a significant increase in the seeding efficiency of BMSCs (p = 0.027). In the calvarial defects implanted with BMSC-seeded poly(LLA-co-CL)/nDPs scaffolds, live imaging at 12 weeks discloses a significant increase in osteogenic metabolic activity (p = 0.016). Microcomputed tomography, confirmed by histological data, reveals a substantial increase in bone volume (p = 0.021). The results show that compared with conventional poly(LLA-co-CL) scaffolds those functionalized with nDPs promote osteogenic metabolic activity and mineralization capacity. It is concluded that poly(LLA-co-CL) composite matrices functionalized with nDPs enhance osteoconductivity and therefore warrant further study as potential scaffolding material for bone tissue engineering.

Functionalization of bone implants with nanodiamond particles and angiopoietin-1 to improve vascularization and bone regeneration

One of the major challenges in bone tissue engineering is adequate vascularization within bone substituents for nutrients and oxygen supply. In this study, the production and results of a new, highly functional bone construct consisting of a commercial three-dimensional β-tricalcium phosphate scaffold (β-TCP, ChronOS®) and hydrophilic, functionalized nanodiamond (ND) particles are reported. A 30-fold increase in the active surface area of the ChronOS + ND scaffold was achieved after modification with ND. In addition, immobilization of angiopoietin-1 (Ang-1) via physisorption within the β-TCP + ND scaffold retained the bioactivity of the growth factor. Homogeneous distribution of the ND and Ang-1 within the core of the three-dimensional scaffold was confirmed using ND covalently labelled with Oregon Green. The biological responses of the β-TCP + ND scaffold with and without Ang-1 were studied in a sheep calvaria critical size defect model showing that the β-TCP + ND scaffold improved the blood vessel ingrowth and the β-TCP + ND + ND + Ang-1 scaffold further promoted vascularization and new bone formation. The results demonstrate that the modification of scaffolds with tailored diamond nanoparticles is a valuable method for improving the characteristics of bone implants and enables new approaches in bone tissue engineering.