Penetration Of Blood Vessels Research Articles

Periosteum-bone inspired hierarchical scaffold with endogenous piezoelectricity for neuro-vascularized bone regeneration

The development of scaffolds for repairing critical-sized bone defects heavily relies on establishing a neuro-vascularized network for proper penetration of nerves and blood vessels. Despite significant advancements in using artificial bone-like scaffolds infused with various agents, challenges remain. Natural bone tissue consists of a porous bone matrix surrounded by a neuro-vascularized periosteum, with unique piezoelectric properties essential for bone growth. Drawing inspiration from this assembly, we developed a periosteum-bone-mimicking bilayer scaffold with piezoelectric properties for regeneration of critical-sized bone defects. The periosteum-like layer of this scaffold features a double network hydrogel composed of chelated alginate, gelatin methacrylate, and sintered whitlockite nanoparticles, emulating the viscoelastic and piezoelectric properties of the natural periosteum. The bone-like layer is composed of a porous structure of chitosan and bioactive hydroxyapatite created through a biomineralization process. Unlike conventional bone-like scaffolds, this bioinspired bilayer scaffold significantly enhances osteogenesis, angiogenesis, and neurogenesis combined with low-intensity pulsed ultrasound-assisted piezoelectric stimulation. Such a scheme enhances neuro-vascularized bone regeneration in vivo. The results suggest that the bilayer scaffold could serve as an effective self-powered electrical stimulator to expedite bone regeneration under dynamic physical stimulation.

Comparative evaluation of polymethylmethacrylate and composite bone cements. Review of the experimental studies results

Current article is a review of experimental studies of different bone cements types and their combinations. Providing of bone fragments stable fixation at osteosynthesis in cases of difficult multifragmental fractures, arthroplasties and other implants using especially in the osteoporosis conditions is a main task of orthopaedic surgery procedures. Polymethylmethacrylate (PMMA) is the first material that is answered to these requirements. The evolution of bone cements resulted in creation of a new composite substance — combination of PMMA and β-threecalciumphosfates (β-TCPh). Combination of these two components allowed to provide high bioabsorbal, osteoconductive and osteointegrative properties along with sufficient durability. In the analyzed works the properties of composite cement CalCemex were evaluated in vivo experiment. It was found that in the case of PMMA penetration of bone tissue into the polymer structure did not occur. Under the conditions of using bone cement with β-TCF admixture, the formation of bone tissue was observed not only on the surface of the implant, but also in the external and internal pores. It is the presence of pores in CalCemex that the authors explain the possibility of penetration of cellular elements, blood vessels and bone formation. Moreover, β-TCPh is included into this material and it is bioresorbed by osteoclasts. This leads to the release of calcium and phosphorus ions and, consequently, simplifies the attachment of the newly formed bone to the bone cement. We assume that composite cement like CalCemex type is a promising material for the treatment of various types of fractures and replacement of bone defects. It should be mentioned that research in this area is ongoing and intensive work is underway to synthesize and study the results of clinical application of composite bone cements with maximum bioactive properties that will not only strengthen bone tissue but also perform osteointegrative function. Key words. Bone cement, polymethylmethacrylate, β-threecalciumphosfates, experiment.

Bacterial Invasion of Pulp Blood Vessels in Teeth with Symptomatic Irreversible Pulpitis

IntroductionThis study described the degenerative changes and infection patterns of the pulp tissue associated with symptomatic irreversible pulpitis. MethodsThe material consisted of 32 extracted teeth with untreated deep caries that were clinically and histologically diagnosed with irreversible pulpitis and were part of the histopathologic collection of 1 of the authors. The controls consisted of intact teeth with normal uninflamed pulps and teeth with reversible pulpitis. Teeth were processed for histopathologic and histobacteriologic analyses. ResultsAll teeth with irreversible pulpitis showed areas of severe acute inflammation, necrosis, microabscesses, and bacterial infection in the pulp chamber. These areas were surrounded by a chronic inflammatory infiltrate, and, at the distance, the pulp tissue was often uninflamed. Bacteria were also observed in the areas surrounding the necrotic foci, both as scattered cells through the extravascular space and at varying numbers within the blood vessel lumen. The number of bacteria and the density of the intravascular bacterial aggregations varied considerably. In one third of the cases, bacteria occurred in the lumen of venules in areas at a considerable distance from the necrotic focus in the coronal third of the root. No intravascular bacteria were noted in the middle and apical segments of the canal. No bacteria were found in the pulps of any of the control specimens. ConclusionsBacterial invasion and colonization of necrotic areas were observed in the pulp of all teeth with caries exposure and symptomatic irreversible pulpitis. Bacterial penetration of blood vessels occurred in all cases, suggesting that this may be an important mechanism of spread of bacterial infection through the pulp tissue in an endodontic infection.

Comparison of hyaluronic acid filler ejection pressure with injection force for safe filler injection.

Owing to the increase in the number of medical procedures performed every year, the frequency of filler injection-related complications has also increased. Although slow, gentle injections with low pressure are usually considered to be safe, the differences in ejection pressure during a filler injection remain to be determined. This study aimed to identify the optimal pressure during filler injections and evaluate its capacity to overcome the arterial blood pressure and reflux the filler material. Twelve combinations of four hyaluronic acid (HA) fillers with different rheological properties and three needles of different diameters were assessed to determine the force exerted by the injection model. The ejection forces corresponding to varying injection forces were measured and HA filler ejection pressures were calculated. The highest and lowest injection forces were achieved using 30- and 25-G needles, respectively. In accordance with the expected ejection force, high ejection pressure was achieved by administering the HA filler under a high injection force. Irrespective of the injection force, the ejection pressure was likely to be higher than the vascular pressure at the time of entry into the vessel, rendering the injection dangerous. During filler injection, penetration of blood vessels and intravascular injection can be avoided by approaching the target area gently using a cannula or needle.

Concept Design, Development and Preliminary Physical and Chemical Characterization of Tamoxifen-Guided-Mesoporous Silica Nanoparticles.

Conventional chemotherapies used for breast cancer (BC) treatment are non-selective, attacking both healthy and cancerous cells. Therefore, new technologies that enhance drug efficacy and ameliorate the off-target toxic effects exhibited by currently used anticancer drugs are urgently needed. Here we report the design and synthesis of novel mesoporous silica nanoparticles (MSNs) equipped with the hormonal drug tamoxifen (TAM) to facilitate guidance towards estrogen receptors (ERs) which are upregulated in breast tumours. TAM is linked to the MSNs using a poly-ʟ-histidine (PLH) polymer as a pH-sensitive gatekeeper, to ensure efficient delivery of encapsulated materials within the pores. XRD, HR-TEM, DLS, SEM, FT-IR and BET techniques were used to confirm the successful fabrication of MSNs. The MSNs have a high surface area (>1000 m2/g); and a mean particle size of 150 nm, which is an appropriate size to allow the penetration of premature blood vessels surrounding breast tumours. Successful surface functionalization was supported by FT-IR, XPS and TGA techniques, with a grafting ratio of approximately 29%. The outcomes of this preliminary work could be used as practical building blocks towards future formulations.

Nitric Oxide-Driven Nanomotor for Deep Tissue Penetration and Multidrug Resistance Reversal in Cancer Therapy.

Poor permeation of therapeutic agents and multidrug resistance (MDR) in solid tumors are the two major challenges that lead to the failure of the current chemotherapy methods. Herein, a zero‐waste doxorubicin‐loaded heparin/folic acid/l‐arginine (HFLA‐DOX) nanomotor with motion ability and sustained release of nitric oxide (NO) to achieve deep drug penetration and effective reversal of MDR in cancer chemotherapy is designed. The targeted recognition, penetration of blood vessels, intercellular penetration, special intracellular distribution (escaping from lysosomes and accumulating in Golgi and nucleus), 3D multicellular tumor spheroids (3D MTSs) penetration, degradation of tumor extracellular matrix (ECM), and reversal of MDR based on the synergistic effects of the motion ability and sustained NO release performance of the NO‐driven nanomotors are investigated in detail. Correspondingly, a new chemotherapy mode called recognition‐penetration‐reversal‐elimination is proposed, whose effectiveness is verified by in vitro cellular experiments and in vivo animal tumor model, which can not only provide effective solutions to these challenges encountered in cancer chemotherapy, but also apply to other therapy methods for the special deep‐tissue penetration ability of a therapeutic agent.

Preparation of Lipid Nanovesicle Contrast Material and Its Application in Ultrasound Diagnosis of Breast Cancer Tumor

In this study, a kind of melanin lipid nanovesicle contrast agent (M-lvca) material with good light absorption and melanin doping is proposed. In this contrast material, lipid nanospheres containing melanin are obtained by ether titration. Then, it is prepared by freeze-drying. In the experimental part, the shape, structure, particle size and absorption spectrum of the material are studied. In vitro agarose gel model, the ultrasound/photoacoustic analysis of the contrast agent is carried out to detect the effect of different concentration of contrast agent on phagocytosis of cells. In the diagnosis of breast cancer, the enhancement boundary, the distribution of contrast agent, the enhancement shape, the range change after enhancement, and the penetration of blood vessels are detected. The experimental results show that the contrast agent has the characteristics of microsphere shape, uniform distribution and strong light absorption ability in the wavelength range of 100 nm∼900 nm. In vitro ultrasound imaging/photoacoustic imaging test, the contrast agent has a good imaging ability. With the increase of the concentration, the imaging ability will be further enhanced. In the diagnosis of breast cancer, there is a significant difference in the indexes related to ultrasound diagnosis of breast cancer after adding M-lvca contrast agent (P < 0.05). Only a few indexes such as enhancement intensity and perfusion defect have no significant difference (P > 0.05). In this study, the proposed M-lvca material can improve the imaging effect of related indicators in the early diagnosis of breast cancer, which is helpful to improve the accuracy of breast cancer diagnosis.

Functionalization of Silk Fibers by PDGF and Bioceramics for Bone Tissue Regeneration

Bone regeneration is a complex, well-organized physiological process of bone formation observed during normal fracture healing and involved in continuous remodeling throughout adult life. An ideal medical device for bone regeneration requires interconnected pores within the device to allow for penetration of blood vessels and cells, enabling material biodegradation and bone ingrowth. Additional mandatory characteristics include an excellent resorption rate, a 3D structure similar to natural bone, biocompatibility, and customizability to multiple patient-specific geometries combined with adequate mechanical strength. Therefore, endless silk fibers were spun from native silk solution isolated from silkworm larvae and functionalized with osteoconductive bioceramic materials. In addition, transgenic silkworms were generated to functionalize silk proteins with human platelet-derived growth factor (hPDGF). Both, PDGF-silk and bioceramic modified silk were then assembled into 3D textile implants using an additive manufacturing approach. Textile implants were characterized in terms of porosity, compressive strength, and cyclic load. In addition, osteogenic differentiation of mesenchymal stem cells was evaluated. Silk fiber-based 3D textile implants showed good cytocompatibility and stem cells cultured on bioceramic material functionalized silk implants were differentiating into bone cells. Thus, functionalized 3D interconnected porous textile scaffolds were shown to be promising biomaterials for bone regeneration.

Effect of High Speed Homogenizer Speed on Particle Size of Polylactic Acid

Polylactic acid (PLA) nanoparticles could provide extended release of drugs, reducing the frequency of injection and increasing the compliance diabetic retinopathy patients. This requires nanoparticles with a mean diameter of approximately 200 nm to facilitate penetration of small and narrow blood vessels in the retina. Mean diameter of PLA particles prepared using the emulsification-evaporation method was found to depend linearly on the speed of the high speed homogenizer, based on the data taken in the range of 1,000-10,000 rpm. PLA nanoparticles with a mean particle diameter of 190 nm with a spherical morphology could be obtained using a homogenizer speed of 10,000 rpm, allowing the use of PLA nanoparticles for extended release of drugs in the retina.

Rickets: Historical, Epidemiological, Pathophysiological, and Pathological Perspectives.

Rickets was a common metabolic disease of bone a century ago in Europe, North America, and East Asia (mainly due to vitamin D deficiency) but was largely eradicated in growing children by use of cod liver oil and the introduction of vitamin D fortification of milk in the 1930s in the United States. Vitamin D deficiency (VDD) remains the most common form of metabolic bone disease that is entirely preventable and treatable. Historically, rickets has appeared in sporadic epidemics and, despite the introduction of numerous preventive strategies, VDD has remained a global health problem amongst children. Moreover, developed countries such as Canada, Australia, the United Kingdom, and the United States have not been exempt from this. The radiological and histological features of rickets are both distinctive and characteristic and they reflect the underlying pathophysiological issue of decreased mineralization of bone as a result of VDD. The radiological features include 1) metaphyseal cupping and fraying, 2) poor mineralization of epiphyseal centers, 3) irregular and widened epiphyseal plates, 4) increased distance between the end of shaft and epiphyseal center, 5) cortical spurs at right angles to the metaphysis, 6) coarse trabeculation, and 7) periosteal reactions. Fractures may also be evident. The histological features of rickets reflect the failure of cartilage to mineralize and undergo resorption. This results in 1) disordered proliferation of chondrocytes in the hypertrophic zone secondary to a lack of apoptosis, 2) loss of the columnar arrangement of chondrocytes that results in thickening and disorganization of the hypertrophic zone, 3) tongue-like projections of cartilage that extend into the spongiosa, 4) irregularity of the limit between the proliferative and hypertrophic zones, and 5) penetration of blood vessels into the hypertrophic zone. The case of a premature 3-month-old female infant, born in the winter months in the arctic region of Canada who died from a lobar pneumonia with an incidental finding of radiological and pathological evidence of rickets, is presented. The case is used to review the entity of rickets from historical, pathophysiological, radiological, and histological perspectives.

Colovesical Fistula: A Rare Complication of Diverticulitis in Young Male

Herniation of colonic mucosa through the circular muscles at the point of penetration of blood vessels results in diverticuli formation. It is seen most commonly in the large bowel in sigmoid colon. Common complications of diverticular disease are inflammation and bleeding per rectum and rarely fistula formation which are seen mostly in elderly patients. A case of colovesical fistula with diverticulitis in young male aged 30 years is reported.

Dual growth factor-loaded core-shell polymer microcapsules can promote osteogenesis and angiogenesis

Growth factors (GFs) are very critical in stem cell differentiation and tissue regeneration. Therefore GF delivery carriers have been a major subject in tissue engineering research. In this study, we prepare and optimize core-shell microcapsules (C-S MCs) for dual GF delivery. The C-S MCs, composed of an alginate shell and poly(lactic-co-glycolic) acid (PLGA) core, are fabricated using an electrodropping method via custom-made coaxial needles. They are 198±38 µm in diameter with an average core size of 90±13 µm, and they are fabricated using an alginate concentration of 1% (w/v), an electrical voltage of 11 kV, and an inner syringe flow rate of 50 µL/min. Using this platform, dual GFs, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) are encapsulated in the alginate shell and PLGA core, respectively. In vitro release tests of dual GF-loaded C-S MCs reveal early release of BMP-2, followed by VEGF on a temporal release profile of 28 days. In vitro study of the dual GF-loaded MCs demonstrates their osteogenic activity with preosteoblasts; osteogenic markers (osteocalcin and type I collagen) are upregulated and both calcium content and alkaline phosphatase (ALP) activity also increased. In addition, C-S MCs combined with collagen and preosteoblasts were subcutaneously transplanted to the dorsal region of nude mice for 3 weeks. Analysis of the retrieved constructs exhibits that both osteogenesis and angiogenesis were more active in the group containing dual GF-loaded MCs, along with deep penetration of blood vessels inside the construct, compared to blank MCs or single GF (BMP-2)-loaded MCs. This study proposes a dual GF delivery carrier using C-S MCs and demonstrates the feasibility of C-S MCs in the induction of osteogenesis and angiogenesis.

Long-term morphological evaluation of porous poly-DL-lactic acid for soft tissue augmentation

Soft tissues are important for aesthetic considerations in implant therapy. The purpose of this study was to investigate soft tissue augmentation by using porous poly-DL-lactic acid (PDLLA)shaped as a tablet, with a diameter of5.0 mmand a height of2.0 mm. Porous PDLLA was implanted between the periosteal and epithelial tissues in 25 rats that were sacrificed at 1, 2, 4, 12, and 24 weeks. The average height of the PDLLA scaffolds at approximately 24 weeks was 1.85 ±0.08 mm, and the molecular weight decreased with time. Sinusoidal capillaries at 1 week, connective tissues at 4 weeks, and necrotic tissues at 24 weeks were observed more than other periods. At 24 weeks, the connective tissue surviving in the pores was confirmed to contain blood vessels; therefore, blood vessels are considered to be critical for the survival of soft tissue in scaffold pores. In this study, PDLLA was found to be useful for soft tissue augmentation in the long term. Although the molecular weight of PDLLA decreased with time, the height of the PDLLA scaffolds was preserved. The connective tissue surviving in the pores of the scaffolds at 24 weeks were associated with blood vessels. Further studies are necessary to investigate the optimal scaffold shape and surface characteristics to improve the penetration of blood vessels.

The development of a bioengineered organ germ method

To bioengineer ectodermal organs such as teeth and whisker follicles, we developed a three-dimensional organ-germ culture method. The bioengineered tooth germ generated a structurally correct tooth, after both in vitro organ culture as well as transplantation under a tooth cavity in vivo, showing penetration of blood vessels and nerve fibers. Our method provides a substantial advance in the development of bioengineered organ replacement strategies and regenerative therapies.

Neovaskularisation der Hornhaut

The penetration of blood vessels in the cornea causes a reduction of transparency and as a result reduced vision or even blindness. Experiments were conducted on 120 New Zealand albino rabbits, separated into different groups. Doses of vasculogenic alkalis provoked burning of the corneas. The following methods were used: Split-lamp investigation of the cornea with fluorescein, fluorescein angiography of the corneal neovessels, light and electron microscopy. Left eyes served as a control. Right eyes were treated with heparin and methylprednisolone, cobalt dichloride, prooxygens and antioxidants. Application of heparin and methylprednisolone reversibly inhibited the process of neoangiogenesis but intensified the corneal ulcer. The application of cobalt dichloride stimulated angiogenesis in the cornea. Electron microscopic observations showed the generation of transport pathways through the walls of neovessels. The basement membrane of the neovessels was formed nearly 4 - 5 months after their growth. Fluorescein angiography showed first increased permeability from the walls of the neovessels and, 4 - 5 months later, poor penetration of the fluorescein through the vessels walls. The application of inhibitors of neoangiogenesis decreases the repair processes in corneal lesions with initial neovascularization. The angiogenic factors are distributed by diffusion in the corneal stroma and neovessels initiate from the nearest limbic vessels. With the support of fluorescein angiography we can specify the time of their formation. The process of regression of the neovessels should be investigated.

Weight loading young chicks inhibits bone elongation and promotes growth plate ossification and vascularization

The mechanical stimuli resulting from weight loading play an important role in mature bone remodeling. However, the effect of weight loading on the developmental process in young bones is less well understood. In this work, chicks were loaded with bags weighing 10% of their body weight during their rapid growth phase. The increased load reduced the length and diameter of the long bones. The average width of the bag-loaded group's growth plates was 75 +/- 4% that of the controls, and the plates showed increased mineralization. Northern blot analysis, in situ hybridization, and longitudinal cell counting of mechanically loaded growth plates showed narrowed expression zones of collagen types II and X compared with controls, with no differences between the relative proportions of those areas. An increase in osteopontin (OPN) expression with loading was most pronounced at the bone-cartilage interface. This extended expression overlapped with tartarate-resistant acid phosphatase staining and with the front of the mineralized matrix in the chondro-osseous junction. Moreover, weight loading enhanced the penetration of blood vessels into the growth plates and enhanced the gene expression of the matrix metalloproteinases MMP9 and MMP13 in those growth plates. On the basis of these results, we speculate that the mechanical strain on the chondrocytes in the growth plate causes overexpression of OPN, MMP9, and MMP13. The MMPs enable penetration of the blood vessels, which carry osteoclasts and osteoblasts. OPN recruits the osteoclasts to the cartilage-bone border, thus accelerating cartilage resorption in this zone and subsequent ossification which, in turn, contributes to the observed phenotype of narrower growth plate and shorter bones.

Hepatic steatosis in obese Chinese children.

(1) to determine the prevalence of asymptomatic hepatic steatosis and presumed nonalcoholic steatohepatitis, in our local population of obese Chinese children referred for medical assessment; and (2) to assess the correlation between severity of ultrasonographic hepatic steatosis and degree of obesity, insulin resistance and serum biochemical abnormalities. Cross-sectional study. In total, 84 obese children, 25 girls and 59 boys with median age and body mass index (BMI) of 12.0 years (interquartile range (IR): 9.5-14.0) and 30.3 kg/m(2) (IR: 27.1-33.4), respectively, referred for medical assessment were studied. All subjects underwent physical examination, anthropometric and dual energy X-ray absorptiometry (DEXA) scan measurements and real-time ultrasonographic (US) examination of the liver. Fasting blood samples were collected for the measurement of liver function, hepatitis status, levels of serum glucose and insulin and lipid profile. Degree of fatty infiltration of the liver was graded according to ultrasonic appearance of liver echotexture, liver-diaphragm differentiation in echo amplitude, hepatic echo penetration and clarity of hepatic blood vessels. All recruited subjects had no history of alcohol abuse and tests for Hepatitis B or C virus were negative. Thorough examination showed all of them to be in general good health without signs of chronic liver disease. Hepatic steatosis identified by defined ultrasonic appearances was diagnosed in 65 subjects (77%); 17 girls and 48 boys. The severity of fatty liver was positively related to anthropometric measurements including BMI, waist and hip circumference, subscapular skinfold thickness; insulin resistance markers [QUICKI and homeostasis model assessment (HOMA)], and hypertriglyceridaemia. Multvariate ordinal regression analysis showed that BMI and raised alanine aminotransferase (ALT) were positively associated with fatty liver. Combination of hepatic steatosis with raised ALT (presumptive NASH) was found in 19 subjects (24%). This group of patients had significantly higher waist hip ratio and conicity index compared to those with isolated hepatic steatosis. Boys with presumed NASH were also found to have significantly higher insulin resistance. Nonalcoholic fatty liver disease (NAFLD) was common among our cohort of obese children referred for medical assessment. The prevalence of simple steatosis and presumed NASH was 77 and 24%, respectively. The severity of US steatosis was positively correlated with BMI, raised ALT, insulin resistance and hypertryglyceridaemia. Ultrasonography being noninvasive and readily available could be used for the monitoring of the progression of hepatic steatosis. Further longitudinal studies are required to determine the natural disease progression and the role of insulin resistance and other factors in the pathophysiology of NAFLD.

Reconstruction of mandibular defects with autologous tissue-engineered bone

Purpose Maxillofacial reconstructive procedures often require bone graft harvesting, which results in donor site morbidity; the use of tissue-engineered bone would eliminate this problem. In this study, a novel scaffold design and new fabrication protocol were used to produce autologous tissue-engineered constructs (scaffolds seeded with cells) to reconstruct segmental mandibular defects in a minipig model. Materials and methods Porcine mesenchymal stem cells were isolated from the ilium. They were expanded in culture and seeded onto poly- dl-lactic-coglycolic acid scaffolds. The constructs were placed in a bioreactor and incubated for 10 days in medium and osteogenic supplements. Four full-thickness bony defects (2 × 2 cm) were created in the same pig’s mandible. The constructs (n = 2) were placed into 2 of the defects as autologous grafts. One unseeded scaffold and 1 empty defect served as controls. At 6 weeks postimplantation, the pig was sacrificed, the mandible was harvested, and the grafted sites were evaluated by clinical, radiographic, and histologic methods. Results The construct-implanted defects appeared to be filled with hard tissue resembling bone, whereas controls were filled with fibrous tissue. Radiographically, the tissue-engineered constructs were uniformly radiodense with bone distributed throughout. The interface between native bone and constructs was indistinct. Complete bone ingrowth was not observed in control defects. Osteoblasts, osteocytes, bone trabeculae, and blood vessels were identified throughout the defects implanted with constructs. Conclusion This “proof-of-principle” study indicates that porcine mandibular defects can be successfully reconstructed by in vitro cultured autologous porcine mesenchymal stem cells on a biodegradable polymer scaffold with penetration of bone and blood vessels.

REGULATION OF GELATINASE-A (MMP-2) PRODUCTION BY OVINE INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS GROWN IN ALGINATE BEAD CULTURE BY TRANSFORMING GROWTH FACTOR-β1AND INSULIN LIKE GROWTH FACTOR-I

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming Growth Factor-β1 (TGF-β1) and Insulin-like Growth Factor-1 (βIGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24h, then stimulated with TGF-β1 (0.1 or 10ng/ml) or IGF-I (2 or 50ng/ml) ±Concanavalin A (20μg/ml) for an additional 48h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72kDa) and active (59kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-β1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-β1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-β1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.

Liver Regeneration on Chicken Chorioallantoic Membrane

To explore how the liver regenerates, liver pieces from 15-day-old chicken embryos were grafted onto the chorioallantoic membrane (CAM) of 9-day-old chicken embryos and cultured for 11 days at the longest. The cultured liver pieces were examined histologically. The liver implants were gradually engulfed into the CAM and underwent necrosis of hepatocytes, except in their peripheral areas, during the first 1–4 days after grafting. Surviving cells in the peripheral areas began to proliferate 4 days after grafting. Thereafter, the cells were assembled into normal liver tissues and represented almost all the areas of the implants 9 days after grafting. Only after penetration of blood vessels from CAM did the liver implants enter a phase of rapid regeneration to form well-organized liver tissues. At the early stage of regeneration, the cells at the peripheral areas did not produce albumin, but reproduced it in the regenerated liver tissues, implying that hepatocytes restored their functions that were temporarily lost in the process of regeneration. Thus, we concluded that the liver pieces from 15-day-old chicken embryos had the ability to form normal liver tissues on CAM and that the blood supply played an important role in liver regeneration.