Thor Friis

Structure-property relationships of silk-modified mesoporous bioglass scaffolds

Porous mesopore-bioglass (MBG) scaffolds have been proposed as a new class of bone regeneration materials due to their apatite-formation and drug-delivery properties; however, the materials inherent brittleness and high degradation and surface instability are major disadvantages, which compromise its mechanical strength and cytocompatibility as a biological scaffold. Silk, on the other hand, is a native biomaterial and is well characterized with respect to biocompatibility and tensile strength. In this study we set out to investigate what effects blending silk with MBG had on the physiochemical, drug-delivery and biological properties of MBG scaffolds with a view to bone tissue engineering applications. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were the methods used to analyze the inner microstructure, pore size and morphology, and composition of MBG scaffolds, before and after addition of silk. The effect of silk modification on the mechanical property of MBG scaffolds was determined by testing the compressive strength of the scaffolds and also compressive strength after degradation over time. The drug-delivery potential was evaluated by the release of dexamethasone (DEX) from the scaffolds. Finally, the cytocompatibility of silk-modified scaffolds was investigated by the attachment, morphology, proliferation, differentiation and bone-relative gene expression of bone marrow stromal cells (BMSCs). The results showed that silk modification improved the uniformity and continuity of pore network of MBG scaffolds, and maintained high porosity (94%) and large-pore size (200-400 microm). There was a significant improvement in mechanical strength, mechanical stability, and control of burst release of DEX in silk-modified MBG scaffolds. Silk modification also appeared to provide a better environment for BMSC attachment, spreading, proliferation, and osteogenic differentiation on MBG scaffolds.

The influence of surface microroughness and hydrophilicity of titanium on the up-regulation of TGFβ/BMP signalling in osteoblasts.

The topography of titanium implants has been identified as an important factor affecting the osseointegration of surgically placed dental implants. Further modification to produce a hydrophilic microrough titanium implant surface has been shown to increase osseointegration compared with microrough topology alone. This study aimed to determine possible molecular mechanisms to explain this clinical observation by examining differences in the whole genome mRNA expression profile of primary human osteoblasts in response to sand-blasted acid-etched (SLA) and hydrophilic SLA (modSLA) titanium surfaces. A decrease in osteoblast proliferation associated with the titanium surfaces (modSLA > SLA > control) correlated with an increase in expression of the osteogenic differentiation markers BSPII and osteocalcin. Pathway analysis demonstrated that a number of genes associated with the TGFβ‑BMP signalling cascade (BMP2, BMP6, SP1, CREBBP, RBL2, TBS3, ACVR1 and ZFYVE16) were significantly differentially up-regulated with culture on the modSLA surface. BMP2 was shown to have the largest fold change increase in expression which was subsequently confirmed at the protein level by ELISA. Several other genes associated with the functionally important mechanisms relevant to bone healing, such as Wnt signalling (CTNNA1, FBX4, FZD6), angiogenesis (KDR), osteoclastogenesis (HSF2, MCL1) and proteolysis (HEXB, TPP1), were also differentially regulated. These results suggest that chemical (hydrophilic) modification of the SLA surface may result in more successful osseointegration through BMP signalling.

Multifunctional magnetic mesoporous bioactive glass scaffolds with a hierarchical pore structure.

Hyperthermia and local drug delivery have been proposed as potential therapeutic approaches for bone defects resulting from malignant bone tumors. The development of bioactive materials with magnetic and drug delivery properties may potentially meet this target. The aim of this study was to develop a multifunctional mesoporous bioactive glass (MBG) scaffold system for both hyperthermic and local drug delivery applications. To this end iron (Fe)-containing MBG (Fe-MBG) scaffolds with a hierarchical large pores structure (300-500 μm) and fingerprint-like mesopores (4.5 nm) have been prepared. The effects of Fe on the mesopore structure and physiochemical, magnetic, drug delivery and biological properties of MBG scaffolds have been systematically investigated. The results show that the morphology of the mesopores varied from straight channels to curved fingerprint-like channels after incorporation of Fe into MBG scaffolds. The magnetism of MBG scaffolds can be tailored by controlling the Fe content. Furthermore, the incorporation of Fe into mesoporous MBG glass scaffolds enhanced the mitochondrial activity and the expression of bone-related genes (ALP and OCN) in human bone marrow mesenchymal stem cells (BMSC) attached to the scaffolds. The Fe-MBG scaffolds obtained also possessed high specific surface areas and demonstrated sustained drug delivery. Thus Fe-MBG scaffolds are magnetic, degradable and bioactive. The multifunctionality of Fe-MBG scaffolds suggests that there is great potential for their use in the treatment and regeneration of large-bone defects caused by malignant bone tumors through a combination of hyperthermia, local drug delivery and osteoconductivity.

ERK-1/2 and p38 in the regulation of hypertrophic changes of normal articular cartilage chondrocytes induced by osteoarthritic subchondral osteoblasts

OBJECTIVE Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process. METHODS Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell-cell interactions were monitored by phosphorylated antibodies and relevant inhibitors. RESULTS OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell-cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK-1/2 signal phosphorylation in cocultured ACCs. The ERK-1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up-regulation of hypertrophic genes in ACCs. CONCLUSION The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK-1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.

Osteoarthritic cartilage chondrocytes alter subchondral bone osteoblast differentiation via MAPK signalling pathway involving ERK1/2

Osteoarthritic subchondral bone is characterized by abnormal bone density and enhanced production of bone turnover markers, an indication of osteoblast dysfunction. Several studies have proposed that pathological changes in articular cartilage influence the subchondral bone changes, which are typical of the progression of osteoarthritis; however, direct evidence of this has yet to be reported. The aim of the present study was to investigate what effects articular cartilage cells, isolated from normal and osteoarthritic joints, may have on the subchondral bone osteoblast phenotype, and also the potential involvement of the mitogen activated protein kinase (MAPK) signalling pathway during this process. Our results suggest that chondrocytes isolated from a normal joint inhibited osteoblast differentiation, whereas chondrocytes isolated from an osteoarthritic joint enhanced osteoblast differentiation, both via a direct and indirect cell interaction mechanisms. Furthermore, the interaction of subchondral bone osteoblasts with osteoarthritic chondrocyte conditioned media appeared to significantly activate ERK1/2 phosphorylation. On the other hand, conditioned media from normal articular chondrocytes did not affect ERK1/2 phosphorylation. Inhibition of the MAPK-ERK1/2 pathways reversed the phenotype changes of subchondral bone osteoblast, which would otherwise be induced by the conditioned media from osteoarthritic chondrocytes. In conclusion, our findings provide evidence that osteoarthritic chondrocytes affect subchondral bone osteoblast metabolism via an ERK1/2 dependent pathway.

Ovine bone- and marrow-derived progenitor cells and their potential for scaffold-based bone tissue engineering applications in vitro and in vivo.

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) and osteoblasts (OB) for clinical use in bone engineering. Prior to clinical application, cell based treatment concepts need to be evaluated in preclinical, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, only sheep aged > 6 years show secondary osteon formation characteristic of human bone. Osteogenic cells isolated from animals of this age group remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than OB derived from tibial compact bone as assessed in standard 2D cultures. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14−/CD31−/CD45−/CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical bone related markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed > 90%. Cells displayed a spindle‐like morphology and formed interconnected networks. In contrast, when implanted subcutaneously into NOD/SCID mice, MPC presented a lower osteogenic potential than OB. In summary, this study provides a detailed characterisation of ovine MPC and OB from a bone engineering perspective and suggests that MPC and OB provide promising means for future bone disease related treatment applications. Copyright

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (L-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (L-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (L-lactide)-block-poly (L-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Expression of chondromodulin‐1 in the temporomandibular joint condylar cartilage and disc

BACKGROUND The temporomandibular joint (TMJ) cartilage consists of condylar cartilage and disc and undergoes continuous remodeling throughout post-natal life. To maintain the integrity of the TMJ cartilage, anti-angiogenic factors play an important role during the remodeling process. In this study, we investigated the expression of the anti-angiogenic factor, chondromodulin-1 (ChM-1), in TMJ cartilage and evaluate its potential role in TMJ remodeling. METHODS Eight TMJ specimens were collected from six 4-month-old Japanese white rabbits. Safranin-O staining was performed to determine proteoglycan content. ChM-1 expression in TMJ condylar cartilage and disc was determined by immunohistochemistry. Three human perforated disc tissue samples were collected for investigation of ChM-1 and vascular endothelial growth factor (VEGF) distribution in perforated TMJ disc. RESULTS Safranin-O stained weakly in TMJ compared with tibial articular and epiphyseal cartilage. In TMJ, ChM-1 was expressed in the proliferative and hypertrophic zone of condylar cartilage and chondrocyte-like cells in the disc. No expression of ChM-1 was observed in osteoblasts and subchondral bone. ChM-1 and VEGF were both similarly expressed in perforated disc tissues. CONCLUSIONS ChM-1 may play a role in the regulation of TMJ remodeling by preventing blood vessel invasion of the cartilage, thereby maintaining condylar cartilage and disc integrity.

Mesodermal and neural crest derived ovine tibial and mandibular osteoblasts display distinct molecular differences

Mandibular osteoblasts originate from the neural crest and deposit bone intramembranously, mesoderm derived tibial osteoblasts by endochondral mechanisms. Bone synthesized by both cell types is identical in structure, yet functional differences between the two cell types may exist. Thus, both matched juvenile and adult mandibular and tibial osteoblasts were studied regarding their proliferative capacity, their osteogenic potential and the expression of osteogenic and origin related marker genes. Juvenile tibial cells proliferated at the highest rate while juvenile mandibular cells exhibited higher ALP activity depositing more mineralized matrix. Expression of Hoxa4 in tibial cells verified their mesodermal origin, whereas very low levels in mandibular cells confirmed their ectodermal descent. Distinct differences in the expression pattern of bone development related genes (collagen type I, osteonectin, osteocalcin, Runx2, MSX1/2, TGF-β1, BAMBI, TWIST1, β-catenin) were found between the different cell types. The distinct dissimilarities in proliferation, alkaline phosphatase activity, the expression of characteristic genes, and mineralization may aid to explain the differences in bone healing time observed in mandibular bone when compared to long bones of the extremities.

Characterization of nano-structural and nano-mechanical properties of osteoarthritic subchondral bone.

BackgroundAlthough articular cartilage is the primary tissues affected by osteoarthritis (OA), the underlying subchondral bone also undergoes noticeable changes. Despite the growing body of research into the biophysical and mechanical properties of OA bone there are few studies that have analysed the structure of the subchondral sclerosis at the nanoscale. In this study, the composition and nano-structural changes of human osteoarthritis (OA) subchondral bone were investigated to better understand the site-specific changes.MethodsOA bone samples were collected from patients undergoing total knee replacement surgery and graded according to disease severity (grade I: mild OA; grade IV: severe OA). Transmission electron microscopy (TEM), Electron Diffraction, and Elemental Analysis techniques were used to explore the cross-banding pattern, nature of mineral phase and orientation of the crystal lattice. Subchondral bone nano-hydroxyapatite powders were prepared and characterised using high resolution transmission electron microscopy (HR-TEM) and fourier transform infrared spectroscopy (FTIR). Subchondal bone mechanical properties were investigated using a nano-indentation method.ResultsIn grade I subchondral bone samples, a regular periodic fibril banding pattern was observed and the c-axis orientation of the apatite crystals was parallel to the long axis of the fibrils. By contrast, in grade IV OA bone samples, the bulk of fibrils formed a random and undulated arrangement accompanied by a circular oriented pattern of apatite crystals. Fibrils in grade IV bone showed non-hierarchical intra-fibrillar mineralization and higher calcium (Ca) to phosphorous (P) (Ca/P) ratios. Grade IV OA bone showed higher crystallinity of the mineral content, increased modulus and hardness compared with grade I OA bone.ConclusionsThe findings from this study suggest that OA subchondral sclerotic bone has an altered mineralization process which results in nano-structural changes of apatite crystals that is likely to account for the compromised mechanical properties of OA subchondral bones.