Hwa-Chang Liu

Gelatin–chondroitin–hyaluronan tri-copolymer scaffold for cartilage tissue engineering

The mechanism by which the cell synthesizes and secretes extracellular matrix (ECM) and is, in turn, regulated by the ECM is termed dynamic reciprocity. The aim of the present work was to produce a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer to mimic natural cartilage matrix for use as a scaffold for cartilage tissue engineering. The scaffold produced had a uniform pore size of about 180 microm and adequate porosity of 75%. Porcine chondrocytes were seeded onto the tri-copolymer scaffold and cultured in Petri dishes or spinner flasks for 2, 3, 4, or 5 weeks. Chondrocytes were uniformly distributed in the scaffold in the spinner flask cultures, but less so in the Petri dish cultures. Secretion of ECM was found under histology examination. In spinner flask cultures, chondrocytes retained their phenotype for at least 5 weeks, as shown immunohistochemically, and synthesized type II collagen. These results show that gelatin/chondroitin sulfate/hyaluronan tri-copolymer has potential for use as a cartilage tissue engineering scaffold.

In vitro effects of low-intensity ultrasound stimulation on the bone cells

Mechanical perturbations serve as extracellular signals to a variety of cells, including bone cells. Low-intensity pulsed ultrasound produces significant multifunctional effects that are directly relevant to bone formation and resorption. Ultrasound stimulation has been shown to accelerate bone-defect healing and trabecular bone regeneration. In this study, we use an in vitro bone cell culture model to investigate the effect of low-intensity pulsed ultrasound. The rat alveolar mononuclear cell-calvaria osteoblast coculture system was used in this study. Before treatment, the bone cells were cultured for 3 days to facilitate their attachment and differentiation. Then, ultrasound exposure (frequency = 1 MHz, intensity = 0.068 W/cm(2)) or sham exposure for 20 min per day was applied until the end of the experiment. Half of the culture media were obtained on the 4th, 5th, 6th, 7th, 8th, 9th, and 10th days for the analysis of cytokines and biochemical parameters. At the end of the experiment, cells were fixed and stained for identification and quantification of the osteoblast and osteoclast cells. After low-intensity pulse ultrasound stimulation, the osteoblast cell counts were significantly increased, whereas the osteoclast cell counts were significantly decreased. The total alkaline phosphatase amount in the culture medium was increased after 7 days of ultrasound stimulation, and tumor necrosis factor-alpha in ultrasound-stimulated bone cells was significantly increased after the 7th day of culture and reached 474.77% of the control medium on the 10th day of culture. The results of this study suggest that low-intensity ultrasound treatment may have a stimulatory effect on bone-healing processes.

Cytokine release from osteoblasts in response to ultrasound stimulation

Bone is a dynamic tissue with a well-balanced homeostasis preserved by both formation and resorption of bone. Normal turnover of bone, however, can be upset by either increased osteoclast activity or decreased osteoblast function; either mechanism alone or both may result in a net loss of bone. Both osteoclasts and osteoblasts could be stimulated by mechanical stimulation in vitro, and it is assumed that this process may occur in vivo as well. In this experiment, we investigated this hypothesis by examining the effects of ultrasound stimulation on osteoblast growth and cytokine release. With this model, we explored the mechanism of low-intensity pulsed ultrasound on osteoblasts growth and upregulation of osteoclasts formation and function by cytokine release. The results showed that specific pulsed ultrasound exposure could enhance osteoblasts population together with increase in TGFbeta1 secretion and decrease in concentration of IL-6 and TNFalpha in the culture medium. Although, animal studies and clinical trial are needed to understand the real process in the whole body, ultrasound stimulation might be a good method for prevention of bone loss due to osteoporosis.

Influence of hydroxyapatite particle size on bone cell activities: an in vitro study.

Over the past decade, a large number of biomaterials have been proposed as artificial bone fillers for repairing bone defects. The material most widely used in clinical medicine is hydroxyapatite. The aim of our investigation was to study the effect of hydroxyapatite size mechanism on osteoblasts. The osteoblasts were cultured in vitro with 0.1% (1 mg/mL) of various sized hydroxyapatite particles (0.5-3.0, 37-63, 177-250, and 420-841 microm) for 1 h, 3 h, 1 day, 3 days, and 7 days. The results showed that adding hydroxyapatite particles to osteoblast cultures can significantly affect osteoblast cell count. Osteoblast populations decreased significantly. Osteoblast mean surface areas also changed significantly. Transforming growth factor-beta1 (TGF-beta1) concentrations in culture medium decreased significantly with the addition of hydroxyapatite particles. Prostaglandin E2 (PGE2) concentrations in medium increased significantly. The changes in TGF-beta1 and PGE2 concentration were more significant and persisted longer in smaller-particle groups. The inhibitory effects of hydroxyapatite particles on osteoblast cell cultures were mediated by the increased synthesis of PGE2. Caution should be exercised before using a hydroxyapatite product which could easily break down into fine particles.

Tissue engineering-based cartilage repair with mesenchymal stem cells in a porcine model†‡

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF‐β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF‐β‐induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF‐β‐induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel‐only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF‐β‐induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects.

Supracondylar fractures of the femur.

Ninety-three supracondylar fractures of the femur treated with 95 degrees angulated condylar plates were retrospectively evaluated. Seventy-one patients were males and 22 were females; ages ranged from 15 to 80 years (mean, 46.9 years). Seventy-two patients were involved in traffic accidents; 44 of them were victims of motorcycle accidents. Twenty patients sustained open fractures. Fifty patients had associated injuries, 41 of them had multiple fractures. There were 13 patients referred for management of complications. The followup periods ranged from 1 to 10 years (4.6 years). Seventy-nine patients had excellent/good results. Eighty-one patients achieved sound union. Stiffness of knee, nonunion, delayed union, and infection were the major complications. Careful selection of the patients, adherence to the principles of anatomic reduction and rigid fixation, and early mobilization are essential for satisfactory outcomes.

The influence of rat mesenchymal stem cell CD44 surface markers on cell growth, fibronectin expression, and cardiomyogenic differentiation on silk fibroin – Hyaluronic acid cardiac patches

Since MSCs contain an abundant of CD44 surface markers, it is of interesting to investigate whether CD44 on rat MSC (rMSCs) influenced cell growth, fibronectin expression and cardiomyogenic differentiation on new SF/HA cardiac patches. For this investigation, we examined the influences of rMSCs with or without a CD44-blockage treatment on the aforementioned issues after they were cultivated, and further induced by 5-aza on SF and SF/HA patches. The results showed that the relative growth rates of rMSCs cultured on cultural wells, SF/HA patches without or with a CD44-blockage treatment were 100%, 208.9+/-7.1 (%) or 48.4+/-6.0 (%) (n=3, for all), respectively, after five days of cultivations. Moreover, rMSCs cultivated on SF/HA patches highly promoted fibronectin expressions (e.g., 1.8x10(5)/cell, in fluorescent intensity) while cells with a CD44-blockage treatment markedly diminished the expressions (e.g., 1.1x10(4)/cell, in fluorescent intensity) on same patches. For investigating possible influences of CD44 surface markers of rMSCs on their cardiomyogenic differentiation, the expressions of specific cardiac genes of cells were examined by using real-time PCR analysis. The results indicated that 5-aza inducing rMSCs significantly promoted the expressions of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions on SF patches and for cells with a CD44-blockage treatment on SF/HA patches. Furthermore, the intensity of the expressions of cardiotin and connexin 43 of 5-aza inducing rMSCs were markedly higher than those of cells with a CD44-blockage treatment after they were cultured on SF/HA patches. Through this study, we reported that CD44 surface markers of rMSCs highly influenced the proliferations, fibronectin expressions and cardiomyogenic differentiation of rMSCs cultivated on cardiac SF/HA patches.

A Novel Mesoporous Biomaterial for Treating Dentin Hypersensitivity

An ideal material has yet to be discovered that can completely treat dentin hypersensitivity; however, calcium phosphate precipitation has exhibited potential value for the treatment of dentin hypersensitivity by the occlusion of dentinal tubules. We hypothesized that a novel mesoporous silica biomaterial (nano CaO@mesoporous silica, NCMS) containing nano-sized calcium oxide particles mixed with 30% phosphoric acid can efficiently occlude dentinal tubules and significantly reduce dentin permeability, even with the presence of pulpal pressure. This highly supersaturated Ca2+-and HPO4 2−ion-containing NCMS paste was brushed onto dentin surfaces, and the ions diffused deeply into the dentinal tubules and formed a CaHPO4·2H2O precipitation with a depth of 100 μm. The results of the dentin permeability tests showed that the novel mesoporous material exhibited a significant reduction in dentin permeability (p < 0.05), even under simulated pulpal pressure, as compared with our previously developed material, DP-bioglass, and a commercial desensitizing material, Seal & Protect®.

Lineage Differentiation‐Associated Loss of Adenoviral Susceptibility and Coxsackie‐Adenovirus Receptor Expression in Human Mesenchymal Stem Cells

Previous reports debated the effects of differentiation on adenoviral vector (AdV) transduction efficiency and Cox‐sackie‐adenovirus receptor (CAR) expression. This prompted us to investigate the efficiency of AdV transduc‐tion and CAR expression in human mesenchymal stem cells (hMSCs) and their differentiated progeny. Current results revealed high efficiency (>90%) of AdV transduction and a consistent level of CAR expression in hMSCs by the use of AdV carrying the enhanced green fluorescent protein reporter gene. Competition of CAR with blocking monoclonal antibody RmcB resulted in a reduction in transduction efficiency, indicating the CAR involvement in transduction of hMSCs. The cells were then induced to differentiate into bone, fat, or neural cells, and results demonstrated that the differentiation was accompanied with a consistent decline in AdV transduction and a decrement in CAR expression. Cells were infected with AdV and then induced into differentiation, and results demonstrated that transduced cells preserved differentiation potentials and still had transgene expression in a subpopulation of cells for 4 weeks and even in tested lineage‐specific differentiation. According to the present investigation, undifferentiated hMSCs can serve as a gene‐delivering system, and gene transfer into hMSCs before differentiation can resolve the difficulties in transduction of their differentiated progeny.

Theoretical study of temperature elevation at muscle'bone interface during ultrasound hyperthermia

This paper examines the distributions of the SAR (specific absorption rate) ratio and temperature elevation when an ultrasound beam propagates through the interface of muscle and bone. This interface is regarded as a flat boundary to partition the energy of the ultrasound beam, and the analytical solution of temperature distribution is based on the steady-state bio-heat transfer equation. The parameters considered are the incident angle of ultrasound beam, the ultrasound frequency, the acoustic attenuation coefficients of refracted longitudinal and shear waves in bone, and the blood perfusion in muscle. The results show that the peak of the SAR ratio is always at the interface of muscle and bone, while the peak of temperature is located in the bone region beyond the interface. A muscle with lower perfusion or a bone with higher acoustic attenuation results in the shifting of the temperature peak closer to the interface. It is more difficult to heat a higher perfused muscle in front of a bone using a lower frequency ultrasound since the temperature elevation for bone relative to muscle is greater.