ABSTRACT
Orthoregeneration is defined as a solution for orthopaedic conditions that harnesses the benefits of biology to improve healing, reduce pain, improve function, and, optimally, provide an environment for tissue regeneration. Options include drugs, surgical intervention, scaffolds, biologics as a product of cells, and physical and electromagnetic stimuli. The goal of regenerative medicine is to enhance the healing of tissue after musculoskeletal injuries as both isolated treatment and adjunct to surgical management, using novel therapies to improve recovery and outcomes. Various orthopaedic biologics (orthobiologics) have been investigated for the treatment of pathology involving the spine, including lower back pain, with or without numbness and/or dysfunction in the lower extremities, disc herniation, spinal stenosis, and spondylolisthesis. Promising and established treatment modalities include repair of the annulus fibrosis, injection of expanded or nonexpanded autologous or allogenic cells that are chondrogenic or from a stem cell lineage used to promote matrix tissue regeneration of the intervertebral disc, including nucleus pulpous cells and mesenchymal stem cells isolated from bone marrow, umbilical cord blood, or adipose tissue; and injection of platelet-rich plasma, platelet-rich fibrin, or fibrin sealant. Early clinical studies show promise for pain reduction and functional recovery. LEVEL OF EVIDENCE: Level V, expert opinion.
Subject(s)
Biological Products , Intervertebral Disc Degeneration , Intervertebral Disc Displacement , Intervertebral Disc , Low Back Pain , Humans , Biological Products/therapeutic use , Intervertebral Disc/pathology , Intervertebral Disc/surgery , Intervertebral Disc Displacement/pathologyABSTRACT
Intervertebral disc herniation is a common spinal disorder that is often treated with discectomy when conservative measures fail. To devise therapeutic strategies for tears in the annulus fibrosus (AF), the regenerative capability of AF cells under spinal loading needs to be addressed. We hypothesized that the compressive loading associated with deformation in AF cells reduces synthetic and degradative activities in extracellular matrix and cell proliferation. We evaluated expression of key matrix molecules and cell proliferation by RT-PCR and immunohistochemistry by inner and outer bovine AF cells incubated under hydrostatic pressure (HP), arc-bending strain (Strain), and combined HP and Strain (HP/Strain) mimicking spinal loading. Inner AF cells showed significantly increased levels of aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase-1, and tissue inhibitor of metalloproteinases-2 by 6 days under HP (p < 0.05), with a tendency toward increased matrix metalloproteinase-13. Outer AF cells demonstrated a significant decline in collagen type-2 under Strain and HP/Strain (p < 0.05) and a tendency toward suppression of collagen type-1 and elastin expression compared to HP and unloaded control. On the other hand, proliferating cell nucleus antigen increased significantly under Strain and HP/Strain in inner AF and declined under unloaded and HP in outer AF (p < 0.05). Immunohistology findings supported reductions in gene expressions of matrix molecules. Thus, changes in HP/Strain in AF appear to diminish synthetic and degradative activities while increasing cell proliferation. To promote regeneration, continuous overloading should be avoided, as it converts the synthetic activity to a state in which tissue repair is limited.
Subject(s)
Annulus Fibrosus , Cell Proliferation , Extracellular Matrix , Hydrostatic Pressure , Animals , Cattle , Annulus Fibrosus/metabolism , Extracellular Matrix/metabolism , Cells, Cultured , Aggrecans/metabolism , Stress, Mechanical , Tissue Inhibitor of Metalloproteinase-2/metabolism , Collagen Type II/metabolismABSTRACT
In reflex cytology, the presence of prominent nucleoli in immature metaplastic squamous cells (IM) may be underdiagnosed due to variations in interpretation. The aim of this study is to identify human papillomaviruses (HPVs) that infect IM clusters in cervical intraepithelial neoplasia 2 (CIN2) on Papanicolaou (Pap) smears to determine the cytological features of lesion-derived cells. Thirty-two patients with a simultaneous diagnosis of CIN2 on biopsy and high-grade squamous intraepithelial lesions (HSIL) on cytology as well as with IM clusters on HSIL smears were included. CIN2 tissues and HSIL and IM clusters on Pap smears were isolated by manual microdissection, and HPV types were identified by PCR-based genotyping. The nuclear area within the IM clusters was also measured. The median nuclear area of HPV-negative IM clusters was 48 µm2 , with a coefficient of variation (CV) of 0.20; those of HPV-positive clusters were 66 µm2 and 0.34, respectively. The cut-off values of the nuclear area and CV for HPV positivity were 62 µm2 and 0.25, respectively. IM clusters composed of cells with a nuclear area of more than twice that of neutrophils or cells with a wide variation in nuclear sizes are likely to be neoplastic cells caused by HPV.
Subject(s)
Carcinoma, Squamous Cell , Papillomavirus Infections , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Female , Humans , Vaginal Smears , Papillomavirus Infections/diagnosis , Papanicolaou Test , Papillomaviridae/genetics , Human Papillomavirus Viruses , Carcinoma, Squamous Cell/pathologyABSTRACT
The oncogenic potential of human papillomavirus (HPV) may be used to determine the tissue tropism of each HPV type. Cervical cancer develops in the squamo-columar junction of the cervices, and most lesions are induced by high-risk (HR) HPV types. This suggests that HR types preferentially infect the cervix, whereas the preferential infection site for low-risk (LR) types is not well defined. The determination of HPV tropism when using cytology samples can be uncertain since it is difficult to avoid contamination of cell samples between the cervix and the vagina. Herein, cell samples were carefully collected by independently scraping the cervix and vagina, after which the HPV types were determined. HPV tissue tropism was determined by considering what HPV types were positive at only one of the sites (the cervix or the vagina) as the viruses that preferentially infected that site. This method revealed that all LR types were only identified in vaginal samples, whereas 87% of HR types were identified in cervical sites. Thus, LR types may preferentially infect the vagina, whereas HR types infect the cervix. These findings suggest that preferential tissue tropism of certain HPV types is a probable factor for malignant progression.
Subject(s)
Papillomavirus Infections , Uterine Cervical Neoplasms , Female , Humans , Human Papillomavirus Viruses , Papillomaviridae , VaginaABSTRACT
STUDY DESIGN: Isolated human nucleus pulposus (hNP) cells from the degenerated intervertebral disc (IVD) were incubated under hydrostatic pressure (HP) and evaluated for regenerative potential. OBJECTIVES: To characterize metabolic turnover in hNP cells isolated from degenerated IVDs classified by Pfirrmann grade under physiologically relevant HP at high osmolality in vitro. SUMMARY OF BACKGROUND DATA: We demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic HP followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells. MATERIALS AND METHODS: hNP cells isolated from discarded tissue classified as Pfirrmann grade 2 to 3 (n = 13: age, 46.7 ± 14.0) and grade 4 (n = 13: age, 53.0 ± 11.5) were incubated under cyclic HP at 0.2 to 0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules and catabolic and anticatabolic proteins were evaluated. RESULTS: Aggrecan and collagen type II expression were significantly more upregulated under HP in grades 2 to 3 than in grade 4 tissues (both, P < 0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in grades 2 to 3, whereas a negative correlation was found in grade 4 ( P < 0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP. CONCLUSIONS: Resident cells in mild-moderate degenerated discs classified as Pfirrmann grade 2 to 3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading. RELEVANCE: This study explored the potential of degenerated remnant nucleus pulposus cells under a physiological environment, possibly leading to establishing strategies for IVD regeneration.
Subject(s)
Intervertebral Disc Degeneration , Intervertebral Disc , Nucleus Pulposus , Female , Humans , Animals , Cattle , Adult , Middle Aged , Nucleus Pulposus/metabolism , Intervertebral Disc/metabolism , Hydrostatic Pressure , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 2/pharmacology , Extracellular Matrix/metabolism , Intervertebral Disc Degeneration/pathologyABSTRACT
OBJECTIVE: Autologous chondrocyte implantation was the first cell-based therapy that used a tissue engineering process to repair cartilage defects. Recently improved approaches and tissue-engineered cell constructs have been developed for growing patient populations. We developed a chondrocyte construct using a collagen gel and sponge scaffold and physicochemical stimuli, implanted with a surgical adhesive. We conducted a proof-of-concept study of these improvements using a cartilage defect model in miniature swine. DESIGN: We implanted the autologous chondrocyte constructs into full-thickness chondral defects in the femoral condyle, compared those results with empty and acellular scaffold controls, and compared implantation techniques with adhesive alone and with partial adhesive with suture. Two weeks after the creation of the defects and implantation of the cellular or acellular constructs, we arthroscopically confirmed that the implanted constructs remained at the chondral defects. We evaluated the regenerated tissue macro- and microscopically 6 months after the cell constructs were implanted. The tissues were stained with Safranin-O and evaluated using Sellers' histology grading system. RESULTS: The defects implanted with processed cell constructs and acellular scaffolds were filled with chondrocyte-like round cells and with nearly normal tissue architecture that were significantly greater degree compared to empty defect control. Even with the adhesive alone and with suture alone, the cell construct was composed of the dense cartilaginous matrix that was found in the implantation using both the sutures and the adhesive. CONCLUSION: Implantation of cell constructs promoted regeneration and integration of articular cartilage at chondral defects in swine by 6 months.
Subject(s)
Cartilage Diseases , Cartilage, Articular , Animals , Cartilage Diseases/pathology , Cartilage, Articular/pathology , Cartilage, Articular/surgery , Chondrocytes , Humans , Swine , Swine, Miniature , Tissue Engineering/methodsABSTRACT
Nucleus pulposus (NP) cells are exposed to changes in hydrostatic pressure (HP) and osmotic pressure within the intervertebral disc. We focused on main disc matrix components, chondroitin sulfate proteoglycan (CSPG) and hyaluronan (HA) to elucidate the capability of augmented CSPG to enhance the anabolism of bovine NP (bNP) cells under repetitive changes in HP at high osmolality. Aggrecan expression with CSPG in the absence of HP was significantly upregulated compared to the no-material control (phosphate buffer saline) under no HP at 3 days, and aggrecan expression with CSPG under HP was significantly higher than the control with HA under HP at 12 days. Collagen type I expression under no HP was significantly lower with CSPG than in controls at 3 days. Although matrix metalloproteinase 13 expression under HP was downregulated compared to no HP, it was significantly greater with HA than the control and CSPG, even under HP. Immunohistology revealed the involvement of mechanoreceptor of transient receptor potential vanilloid-4 activation under HP, suggesting an HP transduction mechanism. Addition of CSPG had anabolic and anti-fibrotic effects on bNP cells during the early culture period under no HP; furthermore, it showed synergy with dynamic HP to increase bNP-cell anabolism at later time points.
Subject(s)
Chondroitin Sulfate Proteoglycans/pharmacology , Hydrostatic Pressure , Intervertebral Disc Degeneration/therapy , Intervertebral Disc/drug effects , Anabolic Agents/pharmacology , Animals , Cattle , Cells, Cultured , Extracellular Matrix/drug effects , Intervertebral Disc/pathology , Intervertebral Disc Degeneration/pathology , Nucleus Pulposus/drug effects , Nucleus Pulposus/pathologyABSTRACT
Intervertebral discs (IVDs) are exposed to changes in physicochemical stresses including hydrostatic and osmotic pressure via diurnal spinal motion. Homeostasis, degeneration, and regeneration in IVDs have been studied using in vitro, ex vivo, and animal models. However, incubation of nucleus pulposus (NP) cells in medium has limited capability to reproduce anabolic turnover and regeneration under physicochemical stresses. We developed a novel pressure/perfusion cell culture system and a semipermeable membrane pouch device for enclosing isolated NP cells for in vitro incubation under physicochemical stresses. We assessed the performance of this system to identify an appropriate stress loading regimen to promote gene expression and consistent accumulation of extracellular matrices by bovine caudal NP cells. Cyclic hydrostatic pressure (HP) for 4 days followed by constant HP for 3 days in high osmolality (HO; 450 mOsm/kg H2O) showed a trend towards upregulated aggrecan expression and dense accumulation of keratan sulfate without gaps by the NP cells. Furthermore, a repetitive regimen of cyclic HP for 2 days followed by constant HP for 1 day in HO (repeated twice) significantly upregulated gene expression of aggrecan (P < .05) compared to no pressure and suppressed matrix metalloproteinase-13 expression (P < .05) at 6 days. Our culture system and pouches will be useful to reproduce physicochemical stresses in NP cells for simulating anabolic, catabolic, and homeostatic turnover under diurnal spinal motion.
ABSTRACT
Intervertebral disc degeneration is a well-known cause of disability, the result of which includes neck and back pain with associated mobility limitations. The purpose of this article is to provide an overview of the known molecular mechanisms through which intervertebral disc degeneration occurs as a result of complex interactions of exogenous and endogenous stressors. This review will focus on some of the identified molecular changes leading to the deterioration of the extracellular matrix of both the annulus fibrosus and nucleus pulposus. In addition, we will provide a summation of our current knowledge supporting the role of associated DNA and intracellular damage, cellular senescence's catabolic effects, oxidative stress, and the cell's inappropriate response to damage in contributing to intervertebral disc degeneration. Our current understanding of the molecular mechanisms through which intervertebral disc degeneration occurs provides us with abundant insight into how physical and chemical changes exacerbate the degenerative process of the entire spine. Furthermore, we will describe some of the related molecular targets and therapies that may contribute to intervertebral repair and regeneration.
ABSTRACT
Autologous chondrocyte implantation is a promising therapy for the treatment of the articular cartilage defects. Recently, we have developed a three-dimensional chondrocyte construct manufactured with a collagen gel/sponge scaffold and cyclic hydrostatic pressure. However, the roles of various mechanical stresses, specifically hydrostatic pressure and deviatoric stress, as well as poststress loading, were unclear on metabolic function in chondrocytes. We hypothesized that hydrostatic pressure and deviatoric stresses each alter individual metabolic characteristics of chondrocytes. We embedded human articular chondrocytes within an agarose hydrogel and applied hydrostatic pressure and/or deviatoric stress individually or simultaneously for 4 days. Subsequently, we kept the cell constructs without stress for an additional 3 days. With hydrostatic pressure and/or deviatoric stress, more cells proliferated significantly than no stress (p < .05) and more cells proliferated near the inner side of the construct than the outer (p < .05). Cartilage specific aggrecan core protein and collagen type II were upregulated significantly after off-loading hydrostatic pressure alone at Day 7 (p < .05). On the other hand, these molecules were upregulated significantly immediately after deviatoric stress alone and combined with hydrostatic pressure at Day 4 (p < .05). Tissue inhibitor of metalloproteinase-2 was upregulated significantly after off-loading hydrostatic pressure alone and combined deviatoric stress at Day 7 (p < .05). Metalloproteinnase-13 was upregulated significantly with deviatoric stress at Day 4 (p < .05) and combined with hydrostatic pressure at Day 4. These results suggest that metabolic functions are regulated by the combination of hydrostatic pressure and deviatoric stress and by the timing of stress loading.
Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Matrix Metalloproteinase 13/biosynthesis , Matrix Metalloproteinase 2/biosynthesis , Stress, Mechanical , Adult , Cartilage, Articular/cytology , Chondrocytes/cytology , Female , Humans , Hydrostatic Pressure , Male , Middle Aged , Time FactorsABSTRACT
Intervertebral disc degeneration is ubiquitous among aging patients, and altered matrix homeostasis is one of the key features of this condition. Physicochemical stresses have a significant impact on matrix homeostasis as they lead to progressive degeneration and may be associated with spinal pain and dysfunction. Thus, it is important to understand the cellular and matrix characteristics of nucleus pulposus in response to these stresses, which include hydrostatic and osmotic pressures during alternate loading conditions. We hypothesized that a combination of changes in hydrostatic pressure and in osmotic pressure that mimic normal, daily spinal stress would stimulate anabolic function, whereas a non-realistic combination of those stresses would stimulate catabolic function in nucleus pulposus cells. We examined the effects of these combined stresses, represented by 12 systematic conditions, on the metabolic activities of enzymatically isolated bovine caudal nucleus pulposus in vitro. We measured the gene expression of extracellular matrix (ECM) molecules and proliferating cell nuclear antigen (PCNA) and evaluated the quality of the matrix and the capability of cell proliferation immunohistologically. Combined cyclic hydrostatic pressure at 0.5 MPa, 0.5 Hz, and high osmotic pressure at 450 mOsm upregulated the aggrecan core protein and collagen type-II gene expression significantly (p < 0.05), and showed trends of upregulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1, matrix metalloproteinase-13, and PCNA. ECM, however, contained empty spaces at a high osmotic pressure with and without hydrostatic pressure. Since ECM has highly specialized physicochemical properties, homeostasis should involve not only phenotypic cellular behavior but also turnover of ECM. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:466-476, 2019.
Subject(s)
Extracellular Matrix/metabolism , Nucleus Pulposus/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Animals , Cattle , Hydrostatic Pressure , Nucleus Pulposus/cytology , Osmotic Pressure , Primary Cell Culture , TailABSTRACT
Articular cartilage is compressed with joint-loading and weight-bearing stresses, followed by a bulging of the tissue during times of off-loading. This loading and off-loading causes changes in water content, and thus alterations in osmotic pressure. Another unique characteristic of articular cartilage is that it has longitudinal depth: surface, middle, and deep zones. Since each zone is composed of unique components of highly negative extracellular matrices, each zone has a different level of osmotic pressure. It was unclear how changes in osmotic pressure affected chondrocyte matrix turnover in specific longitudinal zones. Therefore, we hypothesized that a change in extrinsic osmotic pressure would alter the production of extracellular matrices by zone-specific chondrocytes. We incubated spheroidal cartilage organoids, formed by specific longitudinal depth zone-derived chondrocytes, under different levels of osmotic pressure. We compared the gene expression and the immunohistology of the matrix proteins produced by the zone-specific chondrocytes. We found that high osmotic pressure significantly upregulated the transient expression of aggrecan and collagen type-II by all zone-derived chondrocytes (p < 0.05). At a high osmotic pressure, surface-zone chondrocytes significantly upregulated the expression of collagen type-I (p < 0.05), and middle- and deep-zone chondrocytes significantly upregulated matrix metalloproteinase-13 (p < 0.05). The spheroids, once exposed to high osmotic pressure, accumulated extracellular matrices with empty spaces. Our findings show that chondrocytes have zone-specific turnover of extracellular matrices in response to changes in osmotic pressure.
Subject(s)
Cartilage/cytology , Extracellular Matrix/metabolism , Osmotic Pressure , Aggrecans/genetics , Aggrecans/metabolism , Animals , Cartilage/metabolism , Cattle , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type II/genetics , Collagen Type II/metabolism , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolismABSTRACT
Objective The effects of hydrostatic pressure (HP) on the matrix synthesis by human articular chondrocytes have been reported elsewhere. In order to optimize the production of extracellular matrix, we aimed to clarify the effects of repetitive HP on metabolic function by human articular chondrocytes. Design The human articular chondrocytes were expanded and embedded within a collagen gel/sponge scaffold. We incubated these constructs with and without HP followed by atmospheric pressure (AP) and repeated the second HP followed by AP over 14 days. Genomic, biochemical, and histological evaluation were performed to compare the effects of each regimen on the constructs. Results The gene expressions of collagen type II and aggrecan core protein were significantly upregulated with repetitive HP regimens compared with a single HP or AP by 14 days ( P < 0.01 or 0.05). Matrix metalloptoteinase-13 (MMP-13) in AP was upregulated significantly compared to other HP regimens at day 14 ( P < 0.01). No significant difference was observed in tissue inhibitor of metalloproteinases-II. Immunohistology demonstrated that application of HP (both repetitive and single) promoted the accumulation of specific extracellular matrix and reduced a MMP-13. A single regimen of HP followed by AP significantly increased the amount of sulfated glycosaminoglycan than that of the AP, whereas repetitive HP remained similar level of that of the AP. Conclusions Repetitive HP had a greater effect on anabolic activity by chondrocytes than a single HP regimen, which will be advantageous for producing a matrix-rich cell construct.
Subject(s)
Cartilage/metabolism , Chondrocytes/metabolism , Extracellular Matrix/metabolism , Hydrostatic Pressure/adverse effects , Tissue Engineering/methods , Aggrecans/genetics , Cartilage/injuries , Chondrocytes/cytology , Collagen Type II/genetics , Gene Expression , Glycosaminoglycans/metabolism , Humans , Matrix Metalloproteinase 13/metabolismABSTRACT
Articular cartilage has multiple histologically distinct longitudinal depth zones. Development and pathogenesis occur throughout these zones. Cartilage explants, monolayer cell culture and reconstituted 3-dimensional cell constructs have been used for investigating mechanisms of pathophysiology in articular cartilage. Such models have been insufficient to reproduce zone-dependent cellular characteristics and extracellular matrix (ECM) upon investigation into cartilage development and pathogenesis. Therefore, we defined a chondrocyte spheroid model consistently formed with isolated chondrocytes from longitudinal depth zones without extrinsic materials. This spheroid showed zone-dependent characteristics of size, cartilage-specific ECM (collagen types I and II, aggrecan and keratan sulfate) and gene expressions of anabolic and catabolic molecules (matrix molecules and matrix metalloproteinase-13). In addition, the spheroid model is small enough to maintain the viability of cells and point symmetry to analyze the gradient of diffusive molecules. This spheroid organoid model will be useful to elucidate the mechanism of histogenesis and pathogenesis in articular cartilage.
Subject(s)
Cartilage, Articular/cytology , Organoids/cytology , Spheroids, Cellular/cytology , Animals , Cattle , Cell Count , Cell Separation , Chondrocytes/cytology , Gene Expression Profiling , Immunohistochemistry , Reproducibility of Results , Spheroids, Cellular/metabolismABSTRACT
BACKGROUND: Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology. Recently, molecular profiles have been obtained with extraction of a minimal volume of RNA using fluorescent-tagged quantitative polymerase chain reaction (qPCR), which requires high integrity RNA. However, the agarose interferes considerably with the quantity and quality of the extracted RNA. Moreover, little is known about RNA integrity when the RNA is extracted from cell/agarose construct. Thus, in order to obtain RNA of sufficient integrity, we examined various extraction methods and addressed reproducible methodologies for RNA extraction from cell/agarose constructs using spectrophotometry and microfluidic capillary electrophoresis. RESULTS: With various extraction methods using a mono-phasic solution of phenol and guanidine isothiocyanate, we evaluated quantity and quality of total RNA from cell/agarose construct. Extraction with solution of phenol and guanidine isothiocyanate followed by a silica based membrane filter column gave sufficient RNA integrity number, which allowed us to proceed to fluorescent-tagged qPCR for evaluating various cellular activities. CONCLUSIONS: The RNA extraction methods using phenol and guanidine isothiocyanate solution and a silica membrane column can be useful for obtaining high integrity RNA from cell/agarose constructs rich in polysaccharide and extracellular matrix. Our study contributes to further investigation using agarose hydrogels and other materials rich in polysaccharide in the field of cellular and tissue engineering.
Subject(s)
Chondrocytes/metabolism , RNA/analysis , RNA/isolation & purification , Sepharose , Animals , Cattle , Cells, Cultured , Electrophoresis, Capillary , Guanidines , Isothiocyanates , Microfluidic Analytical Techniques , Molecular Biology/methods , Phenol , Reproducibility of Results , SpectrophotometryABSTRACT
BACKGROUND: The chondrogenic potential of adipose-derived stem cells (ASCs) has been previously demonstrated, although several reports have indicated that ASCs produce less cartilage-specific matrix than bone marrow-derived mesenchymal stem cells. In this study, we intended to improve chondrogenic phenotypes of ASCs using hydrostatic pressure (HP), without utilizing any growth factors other than the transforming growth factor-ß1. METHODS: Human ASCs (CD13(+), 44(+), 90(+), 14(-), 31(-), 34(-)) were harvested and cultured. After three passages, the cells were suspended in 0.3% neutralized collagen type I solution and injected into semipermeable membrane tubes, from which 66 pouches were constructed. After a day of incubation, the 66 pouches were divided into three groups. Group HP1: Pouches were incubated for 1 week with treatment of cyclic HP at 0-0.5 MPa (4.93 atm), 0.5 Hz, with a medium replenishment rate of 0.1 mL/min at 37°C, 3% O2, and 5% CO2 in air using a bioprocessor. This was followed by 3 weeks with no HP and without pouches. Group HP2: Pouches were incubated for the first and third week (2 total weeks) with the same condition of Group HP1. No HP was applied in the second and fourth week. Group AP: Pouches with one end opened were incubated without HP. We evaluated the cell constructs histologically and immunohistochemically, as well as for specific gene expression. RESULTS: Accumulation of the matrix in the HP1 and HP2 groups was much denser than AP groups, particularly after 2 weeks. Cell numbers in the HP groups increased gradually in the middle zone and peaked at 1 week after incubation, maintaining their numbers for the entire course on the surface layer of the construct. In the genomic study results, COL 2A1, COL 10A1, ACAN, SOX9, MMP3, and MMP13 were upregulated and COL 1A1, ITGB1, and PCNA were downregulated by HP. There were no significant differences between HP1 and HP2 gene expression. CONCLUSION: It was suggested that HP is especially beneficial in the early stage of chondrogenesis of ASCs. Moreover, the expression profile of genes related to chondrocyte differentiation/proliferation was significantly enhanced by HP loading compared with the AP control.
Subject(s)
Adipose Tissue/cytology , Cartilage/growth & development , Chondrogenesis/drug effects , Collagen/pharmacology , Gels/pharmacology , Hydrostatic Pressure , Stem Cells/cytology , Adult , Cartilage/drug effects , Cell Count , Female , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Stem Cells/drug effects , Stem Cells/metabolismABSTRACT
We tested the effects of mouse genotype (C57BL/6NHsd, NOD/SCID, SAMR1, and SAMP6) and ionizing irradiation on bone wound healing. Unicortical wounds were made in the proximal tibiae, and the time course of spontaneous healing and effects of irradiation were monitored radiographically and histologically. There was reproducible healing beginning with intramedullary osteogenesis, subsequent bone resorption by osteoclasts, gradual bridging of the cortical wound, and re-population of medullary hematopoietic cells. The most rapid wound closure was noted in SAMR1 mice, followed by SAMP6, C57BL/6NHsd, and NOD/SCID. Ionizing irradiation (20 Gy) to the leg significantly delayed bone wound healing in mice of all four genotypes. Mice with genetically-determined predisposition to early osteopenia (SAMP6) or with immune deficiency (NOD/SCID) had impairments in bone wound healing. These mouse models should be valuable for determining the effects of irradiation on bone healing and also for the design and testing of novel bone growth-enhancing drugs and mitigators of ionizing irradiation.
Subject(s)
Bone and Bones/injuries , Genotype , Wound Healing/genetics , Wound Healing/radiation effects , Wounds and Injuries/genetics , Animals , Bone and Bones/pathology , Bone and Bones/radiation effects , Female , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Radiation Dosage , Tibia/injuries , Tibia/pathology , Tibia/radiation effects , Time FactorsABSTRACT
Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, wellcharacterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by threedimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 320 days. The morphology of the TK cells was investigated by phasecontrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and celltoscaffold attachment in the threedimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, threedimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)199, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.
Subject(s)
Cell Culture Techniques/methods , Cell Shape , Cholangiocarcinoma/pathology , Cell Line, Tumor , Cell Membrane Structures/metabolism , Cell Membrane Structures/ultrastructure , Cholangiocarcinoma/ultrastructure , Humans , Tissue ScaffoldsABSTRACT
Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three-dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF-ß pathways. We compared the effects of DBP, TGF-ß1, or BMP-2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT-PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP-2, and likewise DBP effects on Wnt genes and ß-catenin only partially required the TGF-ß pathway, as shown by selective inhibition of TGF-ß/activin receptor-like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP-2 and by TGF-ß1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF-ß pathways.
Subject(s)
Bone Morphogenetic Protein 2/physiology , Signal Transduction/physiology , Transforming Growth Factor beta1/physiology , Wnt Signaling Pathway/drug effects , Animals , Bone Demineralization Technique , Cattle , Chondrogenesis/drug effects , Chondrogenesis/physiology , Fibroblasts/metabolism , Humans , Rats , Wnt Signaling Pathway/physiologyABSTRACT
The addition of cyclic hydrostatic pressure (cHP) to cell culture medium has been used to promote extracellular matrix (ECM) production by articular chondrocytes. Though a combination of cHP followed by atmospheric pressure (AP) has been examined previously, the rationale of such a combination was unclear. We compared the effects of loading once versus twice (combinations of cHP followed by AP) regarding both gene expression and biochemical and histological phenotypes of chondrocytes. Isolated bovine articular chondrocytes were embedded in a collagen gel and incubated for 14 days under conditions combining cHP and AP. The gene expression of aggrecan core protein and collagen type II were upregulated in response to cHP, and those levels were maintained for at least 4 days after cHP treatment. Accumulation of cartilage-specific sulfated glycosaminoglycans following cHP for 7 days and subsequent AP for 7 days was significantly greater than that of the AP control (p < 0.05). Therefore, incubation at AP after loading with cHP was found to beneficially affect ECM accumulation. Manipulating algorithms of cHP combined with AP will be useful in producing autologous chondrocyte-based cell constructs for implantation.