Chia-Ying Lin

The interaction between bone marrow stromal cells and RGD modified three dimensional porous polycaprolactone scaffolds

We previously established a simple method to immobilize the Arg-Gly-Asp (RGD) peptide on polycaprolactone (PCL) two-dimensional film surfaces that significantly improved bone marrow stromal cell (BMSC) adhesion to these films. The current work extends this modification strategy to three-dimensional (3D) PCL scaffolds to investigate BMSC attachment, cellular distribution and cellularity, signal transduction and survival on the modified PCL scaffold compared to those on the untreated ones. The results demonstrated that treatment of 3D PCL scaffold surfaces with 1,6-hexanediamine introduced the amino functional groups onto the porous PCL scaffold homogenously as detected by a ninhydrin staining method. Followed by the cross-linking reaction, RGDC peptide was successfully immobilized on the surface of PCL scaffold. Although the static seeding method used in this study caused heterogeneous cell distribution, the RGD-modified PCL scaffold still demonstrated the improved BMSC attachment and cellular distribution in the scaffold. More importantly, the integrin-mediated signal transduction FAK-PI3K-Akt pathway was significantly up-regulated by RGD modification and a subsequent increase in cell survival and growth was found in the modified scaffold. The present study introduces an easy method to immobilize RGD peptide on the 3D porous PCL scaffold and provides further evidence that modification of 3D PCL scaffolds with RGD peptides elicits specific cellular responses and improves the final cell-biomaterial interaction.

Bone Morphogenetic Proteins and Cancer: Review of the Literature

OBJECTIVEIn addition to their well-known osteogenic properties, bone morphogenetic proteins (BMPs) have developmental and regenerative roles that may impact tumorigenesis and promote tumor spread. Given that the most common site of tumor metastases to bone is the spine, determining whether BMPs can be linked to cancer is of particular relevance to surgeons treating primary or metastatic spinal disease. This article reviews the basic scientific and clinical background of BMPs and their potential role in promoting cancer. METHODSA literature review to identify studies relating to BMP and tumorigenesis was conducted. Databases evaluated included MEDLINE and EMBASE as well as the Cochrane Controlled Trials Register through 2008. RESULTSBone morphogenetic proteins are a diverse class of molecules belonging to the transforming growth factor-β superfamily that serve a variety of biologic functions. Bone morphogenetic proteins have critical roles in stem and progenitor cell biology as regulators of cellular expansion and differentiation. Transforming growth factor-β and related cell signaling pathways as well as stem and progenitor cell signaling have been linked to cancer. Multiple in vitro and in vivo studies suggest a significant role of BMPs in promoting tumorigenesis and metastasis. However, there are also comparable studies that imply that BMPs may have a negative effect on cancer. CONCLUSIONThere is no definitive association between BMPs and the promotion of tumorigenesis or metastasis. However, given the relatively large number of studies reporting a positive effect of BMPs on tumorigenesis or metastasis, the use of BMPs in patients with primary or metastatic spinal tumors should be carefully considered.

Prospective identification of tumorigenic osteosarcoma cancer stem cells in OS99-1 cells based on high aldehyde dehydrogenase activity

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines, of which a significantly higher ALDH activity was present in the OS99‐1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99‐1 cells were grown in NOD/SCID mice, we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDHbr cells) from the OS99‐1 xenografts were much less frequent, averaging 3% of the entire tumor population, compared to those isolated directly from the OS99‐1 cell line. ALDHbr cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDHlo cells), generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDHbr cells illustrated the stem cell characteristics of self‐renewal, the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A, Nanog and Sox‐2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma‐initiating cells and may potentially have therapeutic implications for human osteosarcoma.

Characterization of stem cell attributes in human osteosarcoma cell lines

Osteosarcoma is an aggressive primary bone cancer affecting primarily children and young adolescents. Development of valuable diagnostic indicators and therapeutic agents will be enhanced by the identification and characterization of genes that contribute to its aggressive behavior. Emerging evidence suggests a subpopulation of cancer stem cells within tumors that have been implicated in the pathogenesis of heterogeneous malignant tumors. The purpose of our study was to characterize the stem-like cell population in human osteosarcoma. Four human osteosacoma cell lines were cultured using a previously developed sarcosphere formation assay. The results showed that all human osteosarcoma cell lines possessed an ability to form sarcospheres. More spherical and frequent sarcospheres were observed in OS99-1 and MG63 cells than in Hu09 and Saos-2 cells. Moreover, stem cell markers Oct3/4 and Nanog were examined in osteosarcoma cell lines using RT-PCR and immunological techniques. For the first time, we showed that 2 Oct3/4 isoforms, Oct3/4 A and Oct3/4 B, were expressed in all 4 cell lines although they illustrated different expression patterns. Oct3/4 A was expressed in the nuclei while Oct3/4 B was located in the cytoplasm of a subpopulation of cells found within all four cell lines. Furthermore, elevated expression of Oct3/4 A was seen in the OS99-1, Hu09, and MG63 cells compared to Saos-2 cells while significantly higher expression of Oct3/4 B was detected in Hu09 compared with the other cell lines. In addition, Nanog was detected in a subpopulation of all cell lines, where OS99-1 cells expressed significantly higher levels than Hu09, Saos-2 and MG63 cells. Thus, our data support the cancer stem cell hypothesis, which may have important implications for clinic diagnosis and treatment of osteosarcoma.

Developing consistently reproducible intervertebral disc degeneration at rat caudal spine by using needle puncture: Laboratory investigation

OBJECT The goal in this study was to develop a convenient, less-invasive animal model to monitor progression of intervertebral disc (IVD) degeneration for future testing of new treatments for disc degeneration. METHODS Level 5/6 and 7/8 IVDs of rat caudal spine were stabbed laterally with 18- or 21-gauge hypodermic needles to a depth of 5 mm from the subcutaneous surface with the aid of fluoroscopy. In vivo MR imaging studies were performed at 4, 8, and 12 weeks postsurgery to monitor progression of IVD degeneration. Histological analysis including H & E and safranin O staining, and immunohistochemical studies of collagen type II and bone morphogenetic protein receptor type II (BMPRII) were assessed at 12 weeks postsurgery. RESULTS The 18- and 21-gauge needle-stabbed discs illustrated decreases in both the T2 density and MR imaging index starting at 4 weeks, with no evidence of spontaneous recovery by 12 weeks. Histological staining demonstrated a decreased nucleus pulposus (NP) area, and the NP-anulus fibrosus border became unclear during the progression of disc degeneration. Similar patterns of degenerative signs were also shown in both safranin O- and collagen type II-stained sections. The BMPRII immunohistochemical analysis of stabbed discs demonstrated an increase in BMPRII expression in the remaining NP cells and became stronger in anulus fibrosus with the severity of disc degeneration. CONCLUSIONS After introducing an 18- or 21-gauge needle into the NP area of discs in the rat tail, the stabbed disc showed signs of degeneration in terms of MR imaging and histological outcome measurements. Changes in BMPRII expression in this animal model provide an insight for the effectiveness of delivering BMPs into the region responsible for chondrogenesis for disc repair. This convenient, less-invasive, reproducible, and cost-effective model may be a useful choice for testing novel treatments for disc degeneration.

BMP-2 inhibits the tumorigenicity of cancer stem cells in human osteosarcoma OS99-1 cell line

Previously, based on high ALDH activity, we showed that cancer stem cells (CSCs) could be identified as ALDHbr cells from an aggressive human osteosarcoma OS99-1 cell line. In this study, we evaluate the impact of BMP-2 on CSCs.Three types of BMP receptors were expressed in freshly sorted ALDHbr cells. In vitro, growth of the sorted ALDHbr cells was inhibited by BMP-2. Using RT-PCR analysis, BMP-2 was found to down-regulate the expression of embryonic stem cell markers Oct3/4, Nanog, and Sox-2, and up-regulate the transcription of osteogenic markers Runx-2 and Collagen Type I. In vivo, all animals receiving ALDHbr cells treated with BMP-2 did not form significant tumors, while untreated ALDHbr cells developed large tumor masses in NOD/SCID mice. Immunostaining confirmed few Ki-67 positive cells were present in the sections of tumor containing ALDHbr cells treated with BMP-2. These results suggest that BMP-2 suppresses tumor growth by reducing the gene expression of tumorigenic factors and inducing the differentiation of CSCs in osteosarcoma. BMP-2 or BMP-2-mimetic drugs, if properly delivered to tumor and combined with traditional therapies, may therefore provide a new therapeutic option for treatment of osteosarcoma.

Stress Analysis of the Interface Between Cervical Vertebrae End Plates and the Bryan, Prestige LP, and ProDisc-C Cervical Disc Prostheses : An In Vivo Image-Based Finite Element Study

Study Design. Segmental motion and bone-implant interface stresses were analyzed at C5–C6 levels with Bryan, Prestige LP, and ProDisc-C cervical disc prostheses using an image-based finite element modeling technique. Objective. To predict stress patterns at the interface between prosthesis and lower vertebral end plate to better understand the underlying mechanisms of subsidence and how the load transfer pattern of each disc design affects segmental motion. Summary of Background Data. Subsidence is one of the most commonly reported device-related complications in intervertebral disc arthroplasty. Although clinical outcomes have been reported regarding many types of cervical prostheses, few reports have analyzed the effects of stress from cervical artificial discs to the vertebral end plate. Methods. Three-dimensional voxel finite elements were built for C5–C6 spine unit based on computed tomography images acquired from a patient with indication for cervical disc arthroplasty. Models of facet joints and uncovertebral joints were added and artificial disc designs were placed in the intervertebral disc space. Static analyses were conducted under normal physiologic loads in flexion, extension, and lateral bending with precompression. Results. Bryan disc recovered highest range of motion (4.75°) due to the high elastic nucleus, and therefore imposed the lowest stresses superior to C6. The ProDisc-C and Prestige LP discs caused high stress concentrations around their central fins or teeth, and may initiate bone absorption. Analysis of Prestige LP disc may indicate possible subsidence posteriorly caused by the rear-positioned metal-to-metal joint. Conclusion. Rigidity of the cores (“nuclei”) in Prestige LP and ProDisc-C prostheses guarantee initial maintenance of disc height, but high contact stress takes place at the bone-end plate interface if they are improperly placed or undersized. Anchorage designs add an additional factor that may increase propensity of subsidence, indicated by the high contact stress occurring at the end plate flanges of Prestige LP, and at midline keel fixation on the end plate of ProDisc-C. Although Bryan disc differs in these 2 concerns, it also creates much larger displacement during motion with more variation in disc height that may theoretically increase the load sharing of facet and/or uncovertebral joints compared to more rigid artificial discs.

Analysis of load sharing on uncovertebral and facet joints at the C5-6 level with implantation of the Bryan, Prestige LP, or ProDisc-C cervical disc prosthesis: an in vivo image-based finite element study

OBJECT The goal of this study was to evaluate and compare load sharing of the facet and uncovertebral joints after total cervical disc arthroplasty using 3 different implant designs. METHODS Three-dimensional voxel finite element models were built for the C5-6 spine unit based on CT images acquired from a candidate patient for cervical disc arthroplasty. Models of facet and uncovertebral joints were added and artificial discs were placed in the intervertebral disc space. Finite element analyses were conducted under normal physiological loads for flexion, extension, and lateral bending to evaluate von Mises stresses and strain energy density (SED) levels at the joints. RESULTS The Bryan disc imposed the greatest average stress and SED levels at facet and uncovertebral joints with flexion-extension and lateral bending, while the ProDisc-C and Prestige LP discs transferred less load due to their rigid cores. However, all artificial discs showed increased loads at the joints in lateral bending, which may be attributed to direct impinging contact force. CONCLUSIONS In unconstrained/semiconstrained prostheses with different core rigidity, the shared loads at the joints differ, and greater flexibility may result in greater joint loads. With respect to the 3 artificial discs studied, load sharing of the Bryan disc was highest and was closest to normal load sharing with the facet and uncovertebral joints. The Prestige LP and ProDisc-C carried more load through their rigid core, resulting in decreased load transmission to the facet and uncovertebral joints.

Intradiscal injection of simvastatin retards progression of intervertebral disc degeneration induced by stab injury

IntroductionEarlier work indicates that the cholesterol-lowering drug, simvastatin, is anabolic to chondrogenic expression of rat intervertebral disc (IVD) cells, which suggests a potential role for simvastatin in IVD regeneration. In this study, we expand on our earlier work to test the effectiveness of simvastatin on disc degeneration utilizing a rat tail disc degeneration model.Methods30 rats that underwent 21 G needle-puncture at rat tail discs were injected with simvastatin-loaded poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PEG-PLGA-PEG) gel (5 mg/ml) or vehicle control at 4 weeks after needle injury. All animals were sacrificed 2 weeks after simvastatin injection. Bone morphogenetic protein-2 (BMP-2), aggrecan, collagen type II, and collagen type I messenger ribonucleic acid (mRNA) expression in the rat nucleus pulposus (NP) were measured by real-time polymerase chain reaction (PCR). In vivo magnetic resonance imaging (MRI) was performed to monitor changes in disc degeneration. Rat discs were also assessed by histology using hematoxylin and eosin (H&E) and safranin O staining. In addition, the NP weight, glycosaminoglycan (sGAG) and DNA content were also measured.ResultsA single dose of simvastatin loaded in thermo-sensitive PEG-PLGA-PEG gel injected into the NP had the trend to increase aggrecan expression and sGAG content, and significantly increased mRNA levels of BMP-2, collagen type II, and the differentiation index (the ratio of collagen type II to collagen type I). The decreased NP weight, T2 intensity, as well as MRI index in the rat tail discs induced by needle puncture were significantly reversed after 2 weeks of simvastatin treatment. In addition, simvastatin treatment also improved histological changes induced by needle puncture.ConclusionsA single injection of simvastatin loaded in PEG-PLGA-PEG gel into rat tail discs had the potential to retard or regenerate the degenerative disc.

Simvastatin maintains osteoblastic viability while promoting differentiation by partially regulating the expressions of estrogen receptors α.

BACKGROUND Statin is a specific inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme involved in the cholesterol synthesis pathway. In addition to their long-known efficacy for lowering cholesterol, statins have also been reported to possess anabolic effects on bone. Simvastatin is reported to increase cancellous bone volume, bone formation rate, and cancellous bone compressive strength in vivo. MATERIALS AND METHODS In this report, the effects of simvastatin on osteoprecursor cells were evaluated. The effect on cell viability was determined by MTT assay, whereas differentiation and mineralization were examined using an alkaline phosphatase activity (ALP) test and alizarin red-S staining. Protein expressions related to bone formation, such as estrogen receptor-alpha (ER-α) and beta (ER-β), were evaluated by using a Western blot analysis. To assess whether the osteoinductive effect of simvastatin occurs via estrogen receptor pathway, estrogen receptor agonist (E2) and antagonists (ICI 182,780) were applied to the cultures. RESULTS Cultures grown in the presence of simvastatin exhibited an increased value for ALP activity and mineralization. The results of the Western blot analysis indicated that the addition of simvastatin up-regulated ER-α and ER-β expression with a statistically significant difference in ER-α expression. Treatment of E2 led to an increase of the ALP activity and mineralization, but addition of the estrogen receptor antagonist ICI 182,780 revealed a decrease in both values. CONCLUSIONS Based on these findings, it was concluded that simvastatin could produce positive effects on both the differentiation and mineralization of osteoprecursor cells. Our results also suggested that osteoinductive effects of simvastatin were achieved through ER pathway via the increase of ER-α expression.