Sustained local delivery of insulin for potential improvement of peri-implant bone formation in diabetes

Dental implantation is an effective standard treatment modality to restore missing teeth and maxillofacial defects. However, in diabetics there is an increased risk for implant failure due to impaired peri-implant osseous healing. Early topical insulin treatment was recently shown to normalize diabetic bone healing by rectifying impairments in osteoblastic activities. In this study, insulin/poly(lactic-co-glycolic acid) (PLGA) microspheres were prepared by a double-emulsion solvent evaporation method. Microspheres were then incorporated in fibrin gel to develop a local drug delivery system for diabetic patients requiring implant treatment. In vitro release of insulin from fibrin gel loaded with these microspheres was assessed, and sustained prolonged insulin release over 21 days ascertained. To assess the bioactivity of released insulin and determine whether slow release might improve impaired diabetic bone formation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, mineralized nodule formation, and ELISA (enzyme-linked immunosorbent assay) assays were performed. The insulin released from the drug delivery system stimulated cell growth in previously inhibited cells, and ameliorated the impaired bone-forming ability of human MG-63 cells under high glucose conditions. Fibrin gel loaded with insulin/PLGA microspheres shows potential for improving peri-implant bone formation in diabetic patients.     

Biomechanical models to simulate consequences of maxillofacial surgery

This paper presents the biomechanical finite element models that have been developed in the framework of the computer-assisted maxillofacial surgery. After a brief overview of the continuous elastic modelling method, two models are introduced and their use for computer-assisted applications discussed. The first model deals with orthognathic surgery and aims at predicting the facial consequences of maxillary and mandibular osteotomies. For this, a generic three-dimensional model of the face is automatically adapted to the morphology of the patient by the mean of elastic registration. Qualitative simulations of the consequences of an osteotomy of the mandible can thus be provided. The second model addresses the Sleep Apnoea Syndrome. Its aim is to develop a complete modelling of the interaction between airflow and upper airways walls during breathing. Dynamical simulations of the interaction during a respiratory cycle are computed and compared with observed phenomena.     

A wax barrier to simulate bone resorption for pre-clinical laboratory models of cemented total hip replacements

Pre-clinical tests are often performed to screen new implant designs, surgical techniques, and cement formulations. In this work, we developed a technique to simulate the cement–bone morphology found with postmortem retrieved cemented hip replacements. With this technique, a soy wax barrier is created along the endosteal surface of the bone, prior to cementing of the femoral component. This approach was applied to six fresh frozen human cadaver femora and the resulting cement–bone morphology and micromotion following application of torsional loads were measured on a transverse section of each bone. The contact fraction between cement and bone for the wax barrier specimens (6.4±5.7%, range: 0.5–15%) was similar to that found in postmortem retrievals (10.5±10.3%, range: 0.4–32.5%). Micro-motions at the cement–bone interface for the wax barrier specimens (0.5±1.06 mm, range: 0.005–2.66) were similar, but on average larger than those found with postmortem retrievals (0.092±0.22 mm, range: 0.002–0.73). The use of a wax barrier coating technique could improve experimental pre-clinical tests because it produces a cement–bone interface similar to those of functioning cemented components obtained following in vivo service.     

Mouse models of cardiac chamber formation and congenital heart disease

Congenital heart defects are among the most prevalent and serious birth anomalies in humans. These defects usually involve abnormalities in the formation of specific structures of the developing heart, such as individual chambers or the structures separating one chamber from another. The molecular and cellular pathways responsible for normal heart formation are not well understood, but recent data on single gene knockouts has begun to shed light on the crucial processes that when disrupted result in congenital heart defects.     

The BMP signaling and in vivo bone formation

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smads 1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and their signaling molecules have shown that BMP signaling plays critical roles in bone and cartilage development and postnatal bone formation. BMP activities are regulated at different molecular levels. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue.     

Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo

Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti-CTGF neutralizing antibody caused a dose-dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo.     

In vivo evaluation of bioprinted prevascularized bone tissue

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.     

Numerical simulation of tissue differentiation around loaded titanium implants in a bone chamber

The application of a bone chamber provides a controlled environment for the study of tissue differentiation and bone adaptation. The influence of different mechanical and biological factors on the processes can be measured experimentally. The goal of the present work is to numerically model the process of peri-implant tissue differentiation inside a bone chamber, placed in a rabbit tibia. 2D and 3D models were created of the tissue inside the chamber. A number of loading conditions, corresponding to those applied in the rabbit experiments, were simulated. Fluid velocity and maximal distortional strain were considered as the stimuli that guide the differentiation process of mesenchymal cells into fibroblasts, chondrocytes and osteoblasts. Mesenchymal cells migrate through the chamber from the perforations in the chamber wall. This process is modelled by the diffusion equation. The predicted tissue phenotypes as well as the process of tissue ingrowth into the chamber show a qualitative agreement with the results of the rabbit experiments. Due to the limited number of animal experiments (four) and the observed inter-animal differences, no quantitative comparison could be made. These results however are a strong indication of the feasibility of the implemented theory to predict the mechano-regulation of the differentiation process inside the bone chamber.     

Soft tissue movement and stress shielding do not affect bone ingrowth in the bone conduction chamber

A variety of bone chambers are used in orthopedic research to study bone and tissue ingrowth in small and large animals. If different bone chambers are placed in one species, differences in bone ingrowth are observed. For instance, bone ingrowth in the bone conduction chamber (BCC) is high, but is low or absent in the repeated sampling bone chamber (RSBC). This difference may be explained by the design and fixation of these chambers. It is known that stress shielding and micromovement can influence bone formation. The objective of the study reported here was to determine whether stress shielding or soft tissue movement affected bone ingrowth in the BCC in the goat. Two types of caps were made, with fixation similar to that of the fixation plate of the RSBC. By placing the caps over the BCCs and fixating the caps directly to the tibial bone, the effect of stress shielding was studied. One cap was in direct contact with the bone chamber underneath, the other cap did not touch the chamber. This difference was used to observe whether movement of the soft tissue on top of the chamber and cap would affect bone ingrowth. Each limb received one control chamber without a cap and a chamber with a cap, either with or without contacting the BCC, yielding four implants per goat. After 12 weeks, bone and total tissue ingrowths were measured. Bone ingrowth was seen in 38 of 40 chambers. Total tissue and bone ingrowths were comparable between control chambers and BCCs with a cap, irrespective of type. Neither stress shielding, nor lack of movement of soft tissue affected bone ingrowth. Other factors in the design of the chambers were responsible for the difference in bone ingrowth between the BCC and the RSBC.     

Application of in vivo microdialysis to measure leptin concentrations in adipose tissue

Microdialysis is a relatively new in vivo sampling technique, which allows repeated collecting of interstitial fluid and infusion of effector molecules into the tissue without influencing whole body function. The possibility of using microdialysis catheter with a large-pore size dialysis membrane (100 kDa) to measure concentrations of the adipocyte-derived peptide hormone leptin in interstitial fluid of adipose tissue was explored. Krebs–Henseleit buffer with 40 g/l dextran-70 was used to prevent perfusion fluid loss across the dialysis membrane. The relative recovery of leptin in vitro was determined using CMA/65 microdialysis catheter (100 kDa cut-off, membrane length 30 mm; CMA, Stockholm, Sweden) and four perfusion rates were tested (0.5, 1.0, 2.0, 5.0 μl/min). Furthermore, the microdialysis catheter CMA/65 was inserted into subcutaneous abdominal adipose tissue of 11 healthy human subjects and leptin concentrations in the interstitial fluid of adipose tissue in vivo were measured. The present findings are the first documentation on the use of microdialysis to study local leptin concentrations in the interstitial fluid of adipose tissue.     

Bone tissue formation in organ cultures of human bone marrow

Bone formation in adult human bone marrow organ cultures is described. When culturing marrow fragments, thick bone lamina is formed. It has well-mineralized trabecular bone matrix with bone cells incorporated and is lined with osteoblast-like cells. In cultures of marrow deaggregated cell suspensions thin layers of the bone are only formed. Osteoclast-like cells develop in the cultures.     

Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation

Adeno-associated virus (AAV) is so far the most valuable vehicle for gene therapy because it has no association with immune response and human disease. The present study was conducted to investigate the feasibility of AAV-mediated BMP4 gene transfer for bone formation. In vitro study suggested that AAV-BMP4 vectors could transduce myoblast C2C12 cells and produce osteogenic BMP4. In vivo study demonstrated that new bone formation could be induced by direct injection of AAV-BMP4 into the skeletal muscle of immunocompetent rats. Histological analysis revealed that the newly formed bone was induced through endochondral mechanism. Immunohistochemical staining further demonstrated that AAV-BMP4 gene delivery could mediate long-term transduction, and the involvement of BMP4 expression was responsible for the endochondral ossification. This study is, to our knowledge, the first report in the field of AAV-based BMP gene transfer and should be promising for clinical orthopaedic applications.     

Amylin inhibits bone resorption while the calcitonin receptor controls bone formation in vivo

Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic beta cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/- mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/- mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/- and Amylin +/- mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.     

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.     

Using lazy evaluation to simulate realistic-size repertoires in models of the immune system

We describe a method of implementing efficient computer simulations of immune systems that have a large number of unique B-and/or T-cell clones. The method uses an implementation technique called lazy evaluation to create the illusion that all clones are being simulated, while only actually simulating a much smaller number of clones that can respond to the antigens in the simulation. The method is effective because only 0.001–0.01% of clones can typically be stimulated by an antigen, and because many simulations involve only a small number of distinct antigens. A lazy simulation of a realistic number of clones and 10 distinct antigens is 1000 times faster and 10 000 times smaller than a conventional simulation—making simulations of immune systems with realistic-size repertoires computationally tractable.     

应用拱棚-空调法对极干旱区降水的模拟回收

拱棚法监测表明极干旱区存在潜水蒸发,而土壤温湿度监测表明,土壤水分具备向下运转的条件,这意味着拱棚所监测的水分可能来自降水,极干旱区并不存在深埋潜水蒸发。因此,研究降水的最终去向非常重要。为此笔者应用拱棚-空调法进行对极干旱区降雨模拟回收。结果表明,在极干旱条件下占该区85%以上频次的5 mm的降水经90 d可完全蒸发和回收。回收过程中,棚内地上50 cm的相对湿度(RH)和绝对湿度(AH)分别较棚外增高12.10%和3.50 g/m3,这使降水的回收时间大为延长;另外,土壤内部的温湿度监测表明,洒水后30 cm土壤的温度、RH、AH分别高于棚外对照1.46℃、4.17%和2.50 g/m3,说明有一定数量的降水通过膜下土壤侧向流向了外部,增加了收集时间。回收实验证明极干旱区降水可完全蒸发,该区存在潜水蒸发与GSPAC(Groundwater-Soil-Plant-Atmosphere Continuum)水分的向上运转。