Global chondrocyte gene expression after a single anabolic loading period: Time evolution and re-inducibility of mechano-responses.

Aim of this study was a genome-wide identification of mechano-regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode and characterization of time evolution and re-inducibility of the response. Osteochondral constructs consisting of a chondrocyte-seeded collagen-scaffold connected to ß-tricalcium-phosphate were pre-cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9 × 10 min over 3 hr) before microarray-profiling was performed. Proteoglycan synthesis was determined by 35 S-sulfate-incorporation over 24 hr. Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45-fold, p = 0.016). Among 115 significantly regulated genes, 114 were up-regulated, 48 of them ≥ twofold. AP-1-relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2-phosphorylation and rising MAP3K4 expression. Expression of proteoglycan-synthesizing enzymes CHSY1 and GALNT4 was load-responsive as were factors associated with the MAPK-, TGF-ß-, calcium-, retinoic-acid-, Wnt-, and Notch-signaling pathway which were significantly upregulated SOX9, and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9-signaling showed no consistent regulation. This study associates novel genes with mechanoregulation in chondrocytes, raising SOX9 protein levels with anabolic loading and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback-regulators. Upregulation of mechanosensitive indicators extending differentially into the resting time provides crucial knowledge to maximize cartilage matrix deposition for the generation of high-level cartilage replacement tissue.

Physiological cartilage tissue engineering effect of oxygen and biomechanics.

In vitro engineering of cartilaginous tissues has been studied for many years, and tissue-engineered constructs are sought to be used clinically for treating articular cartilage defects. Even though there is a plethora of studies and data available, no breakthroughs have been achieved yet that allow for implanting in vivo cultured articular cartilaginous tissues in patients. A review of contributions to cartilage tissue engineering over the past decades emphasizes that most of the studies were performed under environmental conditions neglecting the physiological situation. This is specifically pronounced in the use of bioreactor systems which neither allow for application of near physiomechanical stimulations nor for controlling a hypoxic environment as it is experienced in synovial joints. It is suspected that the negligence of these important parameters has slowed down progress and prevented major breakthroughs in the field. This review focuses on the main aspects of cartilage tissue engineering with emphasis on the relation and understanding of employing physiological conditions.

Synthetic calcium phosphate ceramics for treatment of bone fractures.

Bone is a complex natural material with outstanding mechanical properties and remarkable self-healing capabilities. The mechanical strength is achieved by a complex structure of a mineral part comprising apatitic calcium phosphate crystals embedded in an organic matrix. Bone also contains several types of cells constantly replacing mature bone with new bone. These cells are able to seal fractures and fill gaps with new bone in case of structural damage. However, if a defect exceeds a critical size, surgery is necessary to fill the void with a spacer in order to prevent soft tissue from growing into the defect and delaying the healing process. The spacers, also known as bone grafts, can either be made of fresh bone from the patient, of processed bone from donor organisms, or of synthetic materials chemically similar to the mineral part of bone. Synthetic bone void fillers are also known as bone graft substitutes. This review aims at explaining the biological and chemical background that lead to the development of synthetic bone graft substitutes and gives an overview of the current state of development. It also highlights the multidisciplinary nature of biomaterials research, which combines cell biology and medicine with chemistry, mineralogy, crystallography, and mechanical engineering.

The effect of two point mutations in GDF-5 on ectopic bone formation in a beta-tricalciumphosphate scaffold.

The osteoinductivity of human growth-and-differentiation factor-5 (GDF-5) is well established, but a reduced amount of ectopic bone is formed compared to other members of the bone morphogenetic protein (BMP) family like BMP-2. We hypothesized that swap of two BMP-receptor-interacting residues of GDF-5 to amino acids present in BMP-2 (methionine to valine at the sites 453 and 456) may improve the bone formation capacity of the mutant GDF-5. Heterotopic bone formation of a mutant GDF-5 coated beta-TCP carrier was compared to carriers coated with similar amounts (10 microg) of GDF-5 and BMP-2 in SCID mice. Four week explants revealed 6-fold higher ALP activity in the mutant GDF-5 versus the wild type GDF-5 group (p < 0.0001) and 1.4-fold higher levels compared to BMP-2 (p < 0.006). Bone area in histology was significantly higher in mutant GDF-5 versus all other groups at 4 weeks; however, at 8 weeks BMP-2 reached a similar neo-bone formation like mutant GDF-5. Micro-CT evaluation confirmed higher values in the mutant GDF-5 and BMP-2 groups compared to wild type GDF-5. In conclusion, the mutant GDF-5 showed superior bone formation capacity than GDF-5, and a faster induction at similar final outcome as BMP-2. Mutant GDF-5 thus represents a promising new GDF-5 variant for bone regeneration possibly acting via an increased binding affinity to the BMP-type I receptor.

A physical approach to modify the hydraulic reactivity of alpha-tricalcium phosphate powder.

A microsized alpha-tricalcium phosphate (alpha-TCP) powder was calcined at various temperatures (350 degrees C

The effect of platelet-rich plasma on healing in critical-size long-bone defects.

The role of platelet-rich plasma (PRP) as a promoter of bone healing remains controversial. The hypothesis investigated was that PRP improves bone healing of a critical-size diaphyseal radius defect in a rabbit model. The bone defect was filled with a high-surface ceramic scaffold, calcium-deficient hydroxyapatite (CDHA), with the addition of allogenic PRP, mesenchymal stem cells (MSC) or both. PRP yielded better bone formation than the empty CDHA scaffold as determined by both histology and micro-computer tomography (p<0.05) after 16 weeks, whereas no difference was observed on biomechanical testing. Similar behavior was found in samples with MSC; however, the combination of MSC and PRP did not further improve bone healing. Furthermore, the resorption of CDHA was improved by the addition of PRP, MSC and MSC/PRP, but there were no differences between the groups. The areas of bone formation were greater in areas adjacent to the bone resection areas and towards the intact ulna. In conclusion, PRP improves bone healing in a diaphyseal rabbit model on CDHA and the combination of CDHA. This study supports the allogenic use of PRP for bone healing as an off-the-shelf therapy.

Effect of platelet-rich plasma on the in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells on distinct calcium phosphate scaffolds: the specific surface area makes a difference.

The in vitro effect of platelet-rich plasma (PRP) on cell loading, proliferation, and osteogenic differentiation of human mesenchymal stem cells (MSC) is assessed on distinct resorbable and synthetic calcium phosphate scaffolds. A high specific surface area scaffold composed of calcium-deficient hydroxyapatite (CDHA; 48m2/g) is compared with one made out of beta-tricalcium phosphate (beta-TCP; surface area <0.5 m2/g). Fivefold concentrated fresh PRP is applied to scaffolds loaded with 2 x 10(5) MSC (n = 5). These constructs are kept in a medium with osteogenic supplements for 3 weeks. The addition of PRP leads to a higher cell loading efficiency of MSC on CDHA (p = 0.0001), that reaches the values of beta-TCP. Proliferation over 21 days is improved by PRP both on CDHA (p = 0.0001) and beta-TCP (p = 0.014) compared to MSC/calcium phosphate composites. Without the addition of PRP, CDHA has a lower cell loading efficiency (p= 0.0001) and proliferation (p= 0.001) than beta-TCP. The ALP activity is higher in the MSC/ceramics groups than in the monolayer controls (p<0.05). The addition of PRP does not significantly affect ALP activity. However, ALP activity varies considerably within the cell donors and different PRP-pools (p = 0.001), while the cell numbers do not vary within these two parameters. PRP generates a positive effect on the loading efficiency of MSC on the high specific surface scaffold CDHA that thereby reaches the loading efficiency of beta-TCP. PRP improved proliferation, but its osteogenic properties on both calcium phosphate scaffolds are weak.

Porosity and pore size of beta-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: an in vitro and in vivo study.

The interaction of stem cells and ceramics in bone regeneration is still poorly understood. The aim of this study was to examine the influence of the porosity (25%, 65% and 75%) of beta-tricalcium phosphate (TCP) ceramics on osteogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. For the in vitro portion of the study, TCP scaffolds loaded with MSC were kept in osteogenic induction medium for 21 days. For the in vivo portion of the study, scaffolds loaded with undifferentiated MSC were implanted subcutaneously into SCID mice for 8 weeks and compared with similarly implanted controls that were not loaded with MSC. Measurements of total protein as well as specific alkaline phosphatase (ALP) activity were taken as indicators of growth/matrix production and osteogenic differentiation. An increase in the total protein concentration was noted from day 1 to day 21 on the in vitro TCP 65% and TCP 75% scaffolds (p<0.05) with no such increase noted in the TCP 25% specimens. However, the specific alkaline phosphatase activity increased from day 1 to day 21 in all three in vitro specimens (p<0.02) and reached similar levels in each specimen by day 21. In vivo, ALP activity of cell-loaded TCP 65% ceramics was higher when compared with both the TCP 25% and TCP 75% specimens (p<0.046), and higher in the TCP 75% than TCP 25% specimens (p=0.008). Histology revealed mineralization by human cells in the pores of the TCP ceramic scaffolds with a trend toward greater calcification in TCP 65% and 75%. In summary, a higher porosity of TCP scaffolds does not necessarily mean a higher ALP activity in vivo. The distribution and size of the pores, as well as the surface structure, might play an important role for osteogenic differentiation in vivo.

Influence of platelet-rich plasma on osteogenic differentiation of mesenchymal stem cells and ectopic bone formation in calcium phosphate ceramics.

Platelet-rich plasma (PRP) contains a mixture of growth factors that play an important role in wound and fracture healing. While PRP enhanced bone formation by autogenous cancellous bone grafts, its influence in combination with different bone substitutes remained unknown. This study evaluated the effect of PRP on osteogenic differentiation and ectopic bone formation of human mesenchymal stem cells (MSC) in distinct resorbable calcium phosphate ceramics. Calcium-deficient hydroxyapatite (CDHA) blocks with a large specific surface area (48 m2/g) and beta-tricalcium phosphate (beta-TCP) with a low specific surface area (<0.5 m2/g) were loaded with 2 x 10(5) bone marrow-derived MSC. Half of the specimens were treated with 5-fold concentrated PRP. Biocomposites were implanted subcutaneously into SCID mice or kept under osteogenic culture conditions for 2 weeks before implantation. The addition of PRP increased the specific alkaline phosphatase (ALP) activity (p = 0.012) in undifferentiated MSC/CDHA composites but not in MSC/beta-TCP composites. Osteogenic preinduction was ineffective for CDHA and reduced ALP activity of beta-TCP composites significantly at explantation. Ectopic bone formation was stronger in MSC/CDHA (7/32) compared to MSC/beta-TCP (2/30) composites, but no influence of PRP was evident. In conclusion, the effect of PRP depended on the type of ceramic and the differentiation status of the MSC, and enhanced ALP activity of MSC on the high surface scaffold CDHA only, but PRP did not improve osteogenesis in our setting.

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier.

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.