RESUMO
BACKGROUND: Disc cell therapies, in which cells are injected into the degenerate disc in order to regenerate the matrix and restore function, appear to be an attractive, minimally invasive method of treatment. Interest in this area has stimulated research into disc cell biology in particular. However, other important issues, some of which are discussed here, need to be considered if cell-based therapies are to be brought to the clinic. PURPOSE: Firstly, a question which is barely addressed in the literature, is how to identify patients with 'degenerative disc disease' who would benefit from cell therapy. Pain not disc degeneration is the symptom which drives patients to the clinic. Even though there are associations between back pain and disc degeneration, many people with even severely degenerate discs, with herniated discs or with spinal stenosis, are pain-free. It is not possible using currently available techniques to identify whether disc repair or regeneration would remove symptoms or prevent symptoms from occurring in future. Moreover, the repair process in human discs is very slow (years) because of the low cell density which can be supported nutritionally even in healthy human discs. If repair is necessary for relief of symptoms, questions regarding quality of life and rehabilitation during this long process need consideration. Also, some serious technical issues remain. Finding appropriate cell sources and scaffolds have received most attention, but these are not the only issues determining the feasibility of the procedure. There are questions regarding the safety of implanting cells by injection through the annulus whether the nutrient supply to the disc is sufficient to support implanted cells and whether, if cells are able to survive, conditions in a degenerate human disc will allow them to repair the damaged tissue. CONCLUSIONS: If cell therapy for treatment of disc-related disorders is to enter the clinic as a routine treatment, investigations must examine the questions related to patient selection and the feasibility of achieving the desired repair in an acceptable time frame. Few diagnostic tests that examine whether cell therapies are likely to succeed are available at present, but definite exclusion criteria would be evidence of major disc fissures, or disturbance of nutrient pathways as measured by post-contrast MRI.
Assuntos
Condrócitos/transplante , Degeneração do Disco Intervertebral/terapia , Deslocamento do Disco Intervertebral/terapia , Estenose Espinal/terapia , Terapia Baseada em Transplante de Células e Tecidos , Condrócitos/citologia , Humanos , Disco Intervertebral/fisiologia , RegeneraçãoRESUMO
The intervertebral disc is an avascular tissue, maintained by a small population of cells that obtain nutrients mainly by diffusion from capillaries at the disc-vertebral body interface. Loss of this nutrient supply is thought to lead to disc degeneration, but how nutrient supply influences viable cell density is unclear. We investigated two factors that influence nutrient delivery to disc cells and hence cell viability: disc height and blood supply. We used bovine caudal discs as our model as these show a gradation in disc height. We found that although disc height varied twofold from the largest to the smallest disc studied, it had no significant effect on cell density, unlike the situation found in articular cartilage. The density of blood vessels supplying the discs was markedly greater for the largest disc than the smallest disc, as was the density of pores allowing capillary penetration through the bony endplate. Results indicate that changes in blood vessels in the vertebral bodies supplying the disc, as well as changes in endplate architecture appear to influence density of cells in intervertebral discs.
Assuntos
Capilares/anatomia & histologia , Condrócitos/citologia , Disco Intervertebral/irrigação sanguínea , Disco Intervertebral/citologia , Animais , Bovinos , Contagem de Células , Modelos AnimaisRESUMO
PURPOSE: Aggrecan is one of the major macromolecular components of the intervertebral disc (IVD) and its loss is an early sign of degeneration. Restoration of aggrecan, and hence of biomechanical properties, is a major objective of biological therapies. At present, assessment of aggrecan concentration via its glycosaminoglycan (GAG) content is accomplished using biochemical and histological methods which require sacrifice of tissue. A minimally invasive method for assessing GAG, and hence aggrecan, which can avoid destruction of tissue, would be of benefit. METHODS: We have developed a needle micro-osmometer that is capable of measuring flux of saline into excised human nucleus pulposus (NP) tissue. Using the isotropic osmotic stress technique to assess the swelling pressure of the excised NP tissue and assuming negligible collagen tensile stress, we were able to relate the flux to the tissue fixed charge density (FCD). GAG concentration is evaluated from its FCD via the radioactive tracer technique. Samples representing different ages (28-59 years) and degeneration grades (1-4) were analyzed. RESULTS: The flux is controlled by both the osmotic pressure difference across the probe's semi-permeable membrane and by the tissue permeability. A linear correlation was found between flux and the tissue FCD. The equation describing the linear fit is FCD/(total tissue hydration) = 1.97 × 10(-4) + 8283 × flux (R = 0.836, p < 10(-4)). Thus, by measuring saline flux, the concentration of GAG can be determined. CONCLUSIONS: Micro-osmometry provides a reliable and minimally invasive tool for assessing GAG content in excised NP tissue. This method may be usefully applied in tissue engineering applications. It may also be useful for in vivo measurements if the question of the degenerative effect of needle puncture can be overcome.
Assuntos
Glicosaminoglicanos/análise , Degeneração do Disco Intervertebral/fisiopatologia , Disco Intervertebral/química , Osmometria/métodos , Adulto , Agrecanas/metabolismo , Cadáver , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Disco Intervertebral/cirurgia , Degeneração do Disco Intervertebral/metabolismo , Masculino , Pessoa de Meia-Idade , Agulhas , Osmometria/instrumentação , Pressão Osmótica/fisiologia , Reprodutibilidade dos Testes , Fusão VertebralRESUMO
We investigated the distribution of fibrillin-2 and LTBP-2 (latent TGF-ß binding protein-2) in the intervertebral disc of the adult bovine tail. The association of fibrillin-2 and of LTBP-2 with fibrillin-1 was examined by dual immunofluorescence staining. Both fibrillin-2 and LTBP-2 were found extensively distributed in all regions of the disc with the organisation of the network varying significantly region to region. In the outer annulus fibrosus (OAF) both fibrillin-2 and LTBP-2 co-localised with fibrillin-1 forming fibres running parallel to the collagen fibres of the lamellae with the microfibrillar network staining densely in between the adjacent lamellae and also at the boundaries of the collagen bundle compartments. In the inner annulus fibrosus (IAF) and nucleus pulposus (NP), co-localised fibrillin-1,2 and LTBP-2 formed a chondron-like structure around the cell. By contrast, the inter-territorial matrix of the IAF and NP contained a dense network of fibrillin-2 but only sparse/filamentous fibres of fibrillin-1 and LTBP-2. Dual immunostaining revealed that in this region, fibrillin-2 was highly colocalised with elastin. The LTBP-2 network co-localised well with that of fibrillin-1 in all regions and indeed is reported to bind strongly to fibrillin-1. However, interestingly LTBP-2 but not fibrillin-1 or fibrillin-2 was removed by hyaluronidase but not collagenase pre-digestion. Our results suggest that fibrillin-2 and LTBP-2 could play an important role in disc function.
Assuntos
Disco Intervertebral/química , Proteínas de Ligação a TGF-beta Latente/análise , Proteínas dos Microfilamentos/análise , Animais , Bovinos , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Imunofluorescência , Hialuronoglucosaminidase/farmacologia , Proteínas de Ligação a TGF-beta Latente/efeitos dos fármacos , Vértebras Lombares , Proteínas dos Microfilamentos/efeitos dos fármacos , CaudaRESUMO
The elastic network of articular cartilage was investigated by immunohistochemistry using specific antibodies to elastin and fibrillin-1. Articular cartilage was dissected from defined regions of bovine metacarpophalangeal joints. Elastin fibres and microfibrils were dual-immunostained by labelling with distinct fluorescent dyes. A conventional fluorescence microscope combined with a polarized light filter was used to study the organization and degree of colocalization of elastin fibres, microfibrils and of the collagen network. We observed an elaborately organized elastic network. In the uppermost superficial zone, where few cells were present, elastin fibres and microfibrils formed a dense three dimensional network showing some degree of colocalization. The thickness and organization of this elastic network varied dramatically from region to region and was most extensive in the metacarpal palmar region. In the middle and deep zones, very few elastin fibres were observed but microfibrils formed a network in the inter-territorial matrix and dense network around the cells. Our finding of a three dimensional network of dense, well organized elastin fibres and microfibrils in the surface zone of the articular cartilage matrix, and a dense network of microfibrils around the cells deeper into the tissue suggests the elastic network could play both a mechanical and a biological role in articular cartilage.
Assuntos
Cartilagem Articular/ultraestrutura , Colágeno/análise , Elastina/análise , Matriz Extracelular/ultraestrutura , Microfibrilas/ultraestrutura , Animais , Bovinos , Imuno-HistoquímicaRESUMO
Degenerative changes in the disc have long been of interest; they are thought to be strongly associated with low back pain and caused by inappropriate loading or through injury. However, independent of the magnitude of occupational spinal loading, twin studies find that the heritability of lumbar disc degeneration is 34-74%. This finding has led to intensive searches for susceptibility genes; some genes associated with disc degeneration have been identified, though all with small effects on the degenerative process. The complex nature of degenerative changes suggests that many different genes are involved, and that interactions with environmental factors are influential in progression of degeneration. Low back pain itself also appears heritable (30-46%). The most important clinical question though, is not how discs degenerate but is disc degeneration related to low back pain. Imaging studies find many people with degenerate discs or even with discs showing pathological features such as herniations, are asymptomatic. However results are obscured by the lack of consistent definitions of the phenotypes of disc degeneration and of low back pain. Epidemiological studies could help disentangle these complex relationships, but they will only be successful once consistent classifications and phenotypes of both disc degeneration and low back pain are developed.
Assuntos
Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/fisiopatologia , Dor Lombar/genética , Dor Lombar/fisiopatologia , Fenômenos Biomecânicos , Humanos , Suporte de CargaRESUMO
Since tissue-engineered cartilage is avascular, both nutrient supply and metabolic waste removal rely on diffusion. As a result, gradients of nutrients and wastes exist through the construct. Previous models usually calculate gradients of oxygen, glucose, and lactic acid separately, without taking into account the complex interdependence between concentrations of these substrates and rates of metabolism. In this study, these interactions were experimentally examined and incorporated into diffusion models. One-dimensional diffusion-reaction models were developed for three typical culture conditions, that is, static culture, perfusion culture, and suspended culture. The profiles of oxygen, glucose, lactic acid, and pH in the cultured constructs were calculated simultaneously using measured metabolic rates. The maximum construct size and cell density which could be supported before nutrients were depleted in the construct center was identified; a function predicting the relationship between construct dimension and the maximum viable cell density was developed. For constructs incubated under static culture the model demonstrated that the gradients which developed through the medium could not be neglected. Perfusion cultures could support a considerably higher cell density than static cultures, while for batch cultures in a rotating bioreactor, the volume of medium also influences the maximum cell density that could be supported. This study provides useful guidance for design of engineered cartilage constructs.
Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Modelos Biológicos , Engenharia Tecidual , Animais , Reatores Biológicos , Bovinos , Contagem de Células , Técnicas de Cultura de Células , Sobrevivência Celular , Células Cultivadas , Meios de Cultura , Difusão , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Masculino , Consumo de OxigênioRESUMO
Disc degeneration is a common disorder. Although the back pain that can develop in association with this is rarely life-threatening, the annual cost in terms of morbidity, lost productivity, medical expenses and workers' compensation benefits is significant. Surgical intervention as practised currently is directed towards removing the damaged or altered tissue. Development of new treatment modalities is critical as there is a growing consensus that the strategies used currently for symptomatic degenerative disc disease may not be effective. Accordingly, there is a need to develop an entirely new way to treat this disorder; regenerative medicine and tissue engineering approaches appear particularly promising in this regard. This paper reviews some of the challenges that currently are limiting the clinical application of this approach to the treatment of disc degeneration.
Assuntos
Deslocamento do Disco Intervertebral/cirurgia , Disco Intervertebral/cirurgia , Engenharia Tecidual/métodos , Engenharia Tecidual/tendências , Animais , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Transplante de Células/normas , Transplante de Células/tendências , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Humanos , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/fisiopatologia , Avaliação de Resultados em Cuidados de Saúde/métodos , Seleção de Pacientes , Transplante de Células-Tronco/métodos , Transplante de Células-Tronco/tendências , Alicerces Teciduais/normas , Alicerces Teciduais/tendênciasRESUMO
BACKGROUND: Many new treatments for degeneration of the intervertebral disc are being developed which can be delivered through a needle. These require testing in model systems before being used in human patients. Unfortunately, because of differences in anatomy, there are no ideal animal models of disc degeneration. Bovine explant model systems have many advantages but it is not possible to inject any significant volume into an intact disc. Therefore we have attempted to mimic disc degeneration in an explant bovine model via enzymatic digestion. METHODS: Bovine coccygeal discs were incubated with different concentrations of the proteolytic enzymes, trypsin and papain, and maintained in culture for up to 3 weeks. A radio-opaque solution was injected to visualise cavities generated. Degenerative features were monitored histologically and biochemically (water and glycosaminoglycan content, via dimethylmethylene blue). RESULTS AND CONCLUSION: The central region of both papain and trypsin treated discs was macro- and microscopically fragmented, with severe loss of metachromasia. The integrity of the surrounding tissue was mostly in tact with cells in the outer annulus appearing viable. Biochemical analysis demonstrated greatly reduced glycosaminoglycan content in these compared to untreated discs. We have shown that bovine coccygeal discs, treated with proteolytic enzymes can provide a useful in vitro model system for developing and testing potential new treatments of disc degeneration, such as injectable implants or biological therapies.
Assuntos
Modelos Animais de Doenças , Disco Intervertebral/patologia , Técnicas de Cultura de Órgãos , Doenças da Coluna Vertebral/induzido quimicamente , Animais , Bovinos , Meios de Cultura , Glicosaminoglicanos/metabolismo , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/fisiopatologia , Papaína , Radiografia , Doenças da Coluna Vertebral/fisiopatologia , TripsinaRESUMO
In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.
Assuntos
Cartilagem/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Condrócitos/fisiologia , Matriz Extracelular/fisiologia , Sefarose/metabolismo , Engenharia Tecidual/métodos , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Meios de Cultura/química , Líquido Extracelular/química , Géis/metabolismo , Concentração de Íons de Hidrogênio , MasculinoRESUMO
BACKGROUND: Semi-quantitative evaluation of Modic changes (MCs) has recently been proposed as a way to standardise and increase repeatability of clinical studies. This study is aimed at developing semi-quantitative measures of enhancement, given by contrast agent injection, on T1-weighted images in MCs, and to investigate their reliability and relation with MC types. METHODS: Thirty-seven subjects suffering from low back pain underwent T1-weighted and T2-weighted turbo spin-echo sequences. Five minutes after the injection of a paramagnetic contrast agent, a second T1-weighted sequence was acquired. Regions of interest (ROIs) corresponding to MCs were selected manually on the unenhanced image; control ROIs in the "healthy" bone marrow were selected. For each ROI, the mean signal intensity (SI) of unenhanced pixels and the mean absolute and normalised difference in SI between unenhanced and contrast-enhanced pixels values were calculated. RESULTS: A total of 103 MCs were recognised and 61 were semi-quantitatively analysed: 16 type I, 34 type II and 11 type I/II. Regarding controls, MCs I showed a lower SI on the unenhanced T1-weighted images and a marked contrast enhancement (CE); MCs II showed a higher SI than controls on unenhanced images and a lower or comparable CE; and MCs I/II presented an intermediate SI on the unenhanced images and a marked CE. Inter-rater and intra-rater agreements were found to be excellent or substantial. CONCLUSIONS: Semi-quantitative measurements could differentiate MC types in terms of unenhanced SI and of CE with respect to "healthy" bone marrow.
RESUMO
The metabolic environment of disc cells is governed by the avascular nature of the tissue. Because cellular energy metabolism occurs mainly through glycolysis, the disc cells require glucose for survival and produce lactic acid at high rates. Oxygen is also necessary for cellular activity, although not for survival; its pathway of utilization is unclear. Because the tissues are avascular, disc cells depend on the blood supply at the margins of the discs for their nutrients. The nucleus and inner anulus of the disc are supplied by capillaries that arise in the vertebral bodies, penetrate the subchondral bone, and terminate at the bone-disc junction. Small molecules such as glucose and oxygen then reach the cells by diffusion under gradients established by the balance between the rate of transport through the tissue to the cells and the rate of cellular demand. Metabolites such as lactic acid are removed by the reverse pathway. The concentrations of nutrients farthest from the source of supply can thus be low; oxygen concentrations as low as 1% have been measured in the discs of healthy animals. Although gradients cannot be measured easily in humans, they can be calculated. Measured concentrations in surgical patients are in agreement with calculated values.
Assuntos
Metabolismo Energético/fisiologia , Glucose/metabolismo , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Transporte Biológico/fisiologia , Células Cultivadas , Humanos , Oxigênio/metabolismo , Regeneração/fisiologia , Sensibilidade e Especificidade , Doenças da Coluna Vertebral/etiologia , Doenças da Coluna Vertebral/fisiopatologiaRESUMO
At present, the assessment of developing tissue-engineered constructs is almost always carried out destructively using biochemical or histological methods to determine cell number, viability and tissue growth throughout the construct. Since many of these experiments are long, taking weeks or even months to complete, simple and readily applicable non-destructive methods of monitoring changes in cell metabolism, viability and tissue deposition within the construct would be invaluable; such methods could point out adverse responses during the early stages of culture. Here, we describe the use of microdialysis for detecting local changes in cellular metabolism within a tissue-engineered construct. Three-dimensional constructs consisting of bovine articular chondrocytes entrapped in an alginate gel were cultured in a bioreactor for two weeks. Glucose and lactate were monitored by microdialysis, as the major nutrient and metabolite, respectively. Concentration gradients within the construct were evident, with the highest lactate concentrations in the construct centre. The local lactate concentration was a measure of cellular metabolic activity, decreasing as cellular activity fell and increasing as cellular activity was stimulated. Nutrient starvation and cell death in the construct centre could be readily detected in constructs deliberately cultured under adverse conditions. The results show that probe measurements can give an early warning of inappropriate local metabolic changes. Such information during the growth of tissue-engineered constructs would allow either corrective action or else an early end to an unsuccessful test.
Assuntos
Alginatos/química , Condrócitos/fisiologia , Glucose/metabolismo , Ácido Láctico/metabolismo , Engenharia Tecidual/métodos , Animais , Reatores Biológicos , Bovinos , Glucose/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Hidrogéis/química , Ácido Láctico/químicaRESUMO
STUDY DESIGN: A prospective observational study. OBJECTIVE: The aim of this study was to identify the relationship between obesity, quantified by body mass index (BMI), and both back and leg pain in spinal patients. SUMMARY OF BACKGROUND DATA: Obesity and back pain are massive public health problems. Given the poor correlation between pain and a pathological change in the spine, further investigation is required into other, nonpathological predictors such as obesity. METHODS: The Genodisc Study was one of the largest cross-sectional studies of patients presenting to tertiary spinal units and recruited from six centers in four European countries. In total, 2636 patients were recruited over a 5-year period between 2008 and 2013. Both back and leg pain were scored by patients in the range of 0 to 10. Linear regression was used to model the relationship between BMI and pain. Potential confounders included in the model were age, Zung Depression score, episodes of sport, gender, disability benefit, family history, previous surgery, smoking status, work type, clinical diagnosis, and relevant comorbidities. Back and leg pain outcomes were modeled separately. RESULTS: The study included 1160 men and 1349 women with a mean age of 50.9 years and mean BMI of 27.2âkg/m. In our fully adjusted model, a 5-point increase in BMI was associated with greater leg [0.19 units (95% confidence interval 0.08-0.31)] but not back [0.10 units (95% CI -0.02 to 0.22)] pain scores. Although this relationship was statically significant, given the small magnitude of the relationship, the clinical significance is limited. Similarly, female gender, heavy workload, rheumatoid arthritis, previous spine surgery, and depression were associated with higher back and leg pain. CONCLUSION: In this large observational study of spine patients presenting to tertiary European centers, obesity, as measured by increased BMI, was associated with greater leg pain. LEVEL OF EVIDENCE: 2.
Assuntos
Dor nas Costas/complicações , Índice de Massa Corporal , Obesidade/complicações , Dor/complicações , Adulto , Idoso , Dor nas Costas/fisiopatologia , Avaliação da Deficiência , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/fisiopatologia , Dor/fisiopatologia , Medição da Dor , Estudos ProspectivosRESUMO
STUDY DESIGN: Investigation of the elastic network in disc annulus and its function. OBJECTIVE: To investigate the involvement of the elastic network in the structural interconnectivity of the annulus and to examine its possible mechanical role. SUMMARY OF BACKGROUND DATA: The lamellae of the disc are now known to consist of bundles of collagen fibers organized into compartments. There is strong interconnectivity between adjacent compartments and between adjacent lamellae, possibly aided by a translamellar bridging network, containing blood vessels. An elastic network exists across the disc annulus and is particularly dense between the lamellae, and forms crossing bridges within the lamellae. METHODS: Blocks of annulus taken from bovine caudal discs were studied in either their unloaded or radially stretched state then fixed and sectioned, and their structure analyzed optically using immunohistology. RESULTS: An elastic network enclosed the collagen compartments, connecting the compartments with each other and with the elastic network of adjacent lamellae, formed an integrated network across the annulus, linking it together. Stretching experiments demonstrated the mechanical interconnectivities of the elastic fibers and the collagen compartments. CONCLUSION: The annulus can be viewed as a modular structure organized into compartments of collagen bundles enclosed by an elastic sheath. The elastic network of these sheaths is interconnected mechanically across the entire annulus. This organization is also seen in the modular structure of tendon and muscle. The results provide a new understanding annulus structure and its interconnectivity, and contribute to fundamental structural information relevant to disc tissue engineering and mechanical modeling. LEVEL OF EVIDENCE: N/A.
Assuntos
Colágeno/ultraestrutura , Tecido Elástico/ultraestrutura , Disco Intervertebral/ultraestrutura , Microfibrilas/diagnóstico por imagem , Animais , Bovinos , Colágeno/fisiologia , Tecido Elástico/química , Tecido Elástico/fisiologia , Elastina/análise , Fibrilinas , Disco Intervertebral/química , Disco Intervertebral/fisiologia , Microfibrilas/química , Proteínas dos Microfilamentos/análise , Estresse Mecânico , Resistência à Tração , UltrassonografiaRESUMO
The addition of cryopreservative agents (CPAs) to chondrocytes and natural and engineered cartilage is critical to protect the cells and tissues from freezing damage during cryopreservation, but this may cause cell damage, e.g. by osmotic shock. The damage could be minimized by the control of the cell volume excursion with the knowledge of cell membrane permeability. In this study, the cell volume responses of chondrocytes to three commonly used CPAs were evaluated using a perfusion microscope stage. The osmotic response of chondrocytes was measured to the perfusion with 1.4 M dimethyl sulfoxide (Me2SO), 1,2-propanediol and glycerol at 21 degrees C. Cell volumes and their transients were determined with image analysis. The cell membrane permeability parameters, including the hydraulic conductivity (Lp), the CPA permeability (omega) and the reflection coefficients (sigma) in the Kedem-Katchalsky (K-K) model, and the Lp and omega in the two-parameter model were determined. The correlated K-K parameters at 21 degrees C were Lp=0.166 +/- 0.035, 0.149 +/- 0.061, 0.212 +/- 0.041 microm/min atm, omega=(7.630 +/- 0.174) x 10(-2), (1.428 +/- 0.627) x 10(-2), (2.744 +/- 0.775) x 10(-2) microm/s and sigma=0.91 +/- 0.09, 0.82 +/- 0.11, 0.88 +/- 0.10 for Me(2)SO, glycerol and 1,2-propanediol, respectively. For the two-parameter model, the parameter values were Lp=0.163 +/- 0.040, 0.128 +/- 0.031, 0.169 +/- 0.025 microm/min atm, omega=(7.881 +/- 0.178) x 10(-2), (1.529 +/- 0.525) x 10(-2), (3.716 +/- 0.493) x 10(-2) microm/s for Me2SO, glycerol and 1,2-propanediol, respectively. No significant difference in the predictions of cell volume excursion during CPA addition was observed when using either the K-K model or the two-parameter model and it was hence advised to adopt the simple two-parameter model in the evaluation. The measured parameters can be used to optimise the CPA addition and removal protocols to maximize the cell survival during cryopreservation.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Condrócitos/efeitos dos fármacos , Criopreservação/métodos , Dimetil Sulfóxido/farmacologia , Glicerol/farmacologia , Modelos Biológicos , Propilenoglicol/farmacologia , Animais , Bovinos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Temperatura Baixa , Simulação por Computador , Crioprotetores/farmacocinética , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacocinética , Glicerol/farmacocinética , Técnicas In Vitro , Propilenoglicol/farmacocinéticaRESUMO
Articular chondrocytes are exposed to a unique osmotic environment, which varies throughout the depth of cartilage, and in response to mechanical loading or pathological conditions. In light of such osmotic variations we investigated the response of chondrocytes cultured in alginate beads to long term hypo- and hyperosmotic challenge. Following pre-incubation at 380 mOsmol, exposure to hyperosmotic conditions (550 mOsmol) initially decreased 35S-sulphate incorporation, but after 24 hours of culture, rates had recovered and surpassed their original levels. MAP kinase inhibitors abrogated this response suggesting their involvement in the adaptation mechanism. Hypo-osmotic challenge caused a decrease in 35S-sulphate incorporation throughout the culture period. These results suggest that osmolality is a powerful regulator of macromolecular synthesis, and that perturbations in the osmotic environment may alter the set point for turnover.
Assuntos
Adaptação Fisiológica , Cartilagem Articular/citologia , Condrócitos/metabolismo , Alginatos , Animais , Cartilagem Articular/metabolismo , Bovinos , Técnicas de Cultura de Células/métodos , Ácido Glucurônico , Ácidos Hexurônicos , Sistema de Sinalização das MAP Quinases/fisiologia , Microesferas , Concentração Osmolar , Sulfatos/farmacocinéticaRESUMO
Strategies for the biological repair of intervertebral discs derive from the premise that disc degeneration results from impaired cellular activity and, therefore, that these structures can be induced to regenerate by implanting active cells or providing factors that restore normal cellular activity. In vitro and animal studies using this approach have had some success, but whether this success can be reproduced in degenerate human lumbar discs is unknown. Successful repair requires that the disc cells remain viable and active; they therefore need an adequate supply of nutrients. However, as the disc degenerates, the nutrient supply decreases, thereby limiting cell activity and viability. Current biologic approaches might place additional demands on an already precarious nutrient supply. Here, we discuss whether the loss of nutrients associated with disc degeneration limits the effectiveness of biologic approaches, and indicate that this neglected problem requires investigation if clinical application of such therapies is to succeed.
Assuntos
Degeneração do Disco Intervertebral/terapia , Disco Intervertebral/fisiologia , Regeneração , Animais , Fenômenos Fisiológicos Celulares , Humanos , Disco Intervertebral/citologia , Degeneração do Disco Intervertebral/fisiopatologiaRESUMO
The biomechanical behavior of the intervertebral disk ultimately depends on the viability and activity of a small population of resident cells that make and maintain the disk's extracellular matrix. Nutrients that support these cells are supplied by the blood vessels at the disks' margins and diffuse through the matrix of the avascular disk to the cells. This article reviews pathways of nutrient supply to these cells; examines factors that may interrupt these pathways, and discusses consequences for disk cell survival, disk degeneration, and disk repair.