Angiogenic and inflammatory response to biodegradable scaffolds in dorsal skinfold chambers of mice

For tissue engineering, scaffolds should be biocompatible and promote neovascularization. Because little is known on those specific properties, we herein studied in vivo the host angiogenic and inflammatory response after implantation of commonly used scaffold materials. Porous poly(L-lactide-co-glycolide) (PLGA) and collagen-chitosan-hydroxyapatite hydrogel scaffolds were implanted into dorsal skinfold chambers of balb/c mice. Additional animals received cortical bone as an isogeneic, biological implant, while chambers of animals without implants served as controls. Angiogenesis and neovascularization as well as leukocyte-endothelial cell interaction and microvascular permeability were analyzed over 14 day using intravital fluorescence microscopy. PLGA scaffolds showed a slight increase in leukocyte recruitment compared to controls. This was associated with an elevation of microvascular permeability, which was comparable to that observed in isogeneic bone tissue. Of interest, PLGA induced a marked angiogenic response, revealing a density of newly formed capillaries almost similar to that observed in bone implants. Histology showed infiltration of macrophages, probably indicating resorption of the biomaterial. In contrast, hydrogel scaffolds induced a severe inflammation, as indicated by an approximately 15-fold increase of leukocyte-endothelial cell interaction and a marked elevation of microvascular permeability. This was associated by induction of apoptotic cell death within the surrounding tissue and a complete lack of ingrowth of newly formed microvessels. Histology confirmed adequate engraftment of PLGA and isogeneic bone but not hydrogel within the host tissue. PLGA scaffolds show a better biocompatibility than hydrogel scaffolds and promote vascular ingrowth, guaranteeing adequate engraftment within the host tissue.     

Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: an animal model

Biodegradable macroporous cryogels with highly open and interconnected pore structures were produced from dextran modified with oligo L-lactide bearing hydroxyethylmethacrylate (HEMA) end groups in moderately frozen solutions. Tissue responses to these novel scaffolds were evaluated in rats after dorsal subcutaneous implantation, iliac submuscular implantation, auricular implantation, or in calvarial defect model. In no case, either necrosis or foreign body reaction was observed during histological studies. The cryogel scaffolds integrated with the surrounding tissue and the formation of a new tissue were accompanied with significant ingrowth of connective tissue cells and new blood vessels into the cryogel. The tissue responses were significantly lower in auricular and calvarial implantations when compared with the subcutanous and the submuscular implantations. The degradation of the scaffold was slower in bone comparing to soft tissues. The biodegradable cryogels are highly biocompatible and combine extraordinary properties including having soft and elastic nature, open porous structure, and very rapid and controllable swelling. Therefore, the cryogels could be promising candidates for further clinical applications in tissue regeneration.     

A role of histone H4 hypoacetylation in vascular endothelial growth factor expression in colon mucosa adjacent to implanted cancer in athymic mice cecum.

Expression of angiogenic factors is upregulated in hyperplastic mucosa adjacent to colon cancer, and this upregulation is closely associated with cancer growth and metastasis. We investigated the role of histone acetylation in vascular endothelial growth factor (VEGF) expression in hyperplastic mucosa adjacent to orthotopic colon cancer in mice. In the hyperplastic mucosa adjacent to KM12SM tumor in the cecum of athymic mice, VEGF upregulation was associated with hypoxia-inducible factor (HIF)-1alpha induction. The hyperplastic mucosa also showed hypoacetylation of histone H4 and reduction of both p53 and von Hippel-Lindau (VHL) proteins. To examine the effects of growth factors and cytokines on histone acetylation and levels of p53, VHL and HIF-1alpha, the rat intestinal epithelial cell line IEC6 was treated with epidermal growth factor (EGF) and interleukin (IL)-15 for 35 days. Acetylated histone H4, p53 protein and ubiquitinated protein levels were reduced, whereas HIF-1alpha production was upregulated in EGF- and IL-15-treated IEC6 cells. These findings suggest that EGF- or IL-15-induced histone H4 hypoacetylation is associated with repression of p53 and VHL genes in intestinal epithelial cells. The subsequent suppression of protein ubiquitination leads to upregulation of VEGF production by HIF-1alpha retention.     

Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study

Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue. Biphasic implants consisting of pre-fabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements. Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro. Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-L-lactide [P(L)LA], poly-d,l-lactide [P(D,L)LA] or Collagen-hydroxyapatite [Col-HA] and were cultured for 15 weeks. Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation. P(D,L)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape. Little or no breakdown or chondrocyte colonisation was evident in P(L)LA. Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial. These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.     

Tissue engineering of ureteral grafts by seeding urothelial differentiated hADSCs onto biodegradable ureteral scaffolds

The study is aimed to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into urothelial lineage, and to assess possibility of constructing ureteral grafts using the differentiated hADSCs and a novel polylactic acid (PLA)/collagen scaffolds. HADSCs were indirectly cocultured with urothelial cells in a transwell coculture system for urothelial differentiation. After 14 days coculturing, differentiation was evaluated by detecting urothelial lineage markers (cytokeratin-18 and uroplakin 2) in mRNA and protein level. Then the differentiated hADSCs were seeded onto PLA/collagen ureteral scaffolds and cultured in vitro for 1 week. The biocompatibility of the scaffolds was tested by scanning electron microscopy (SEM) and MTT analysis. At last, the cell/scafflod grafts were subcutaneously implanted into 4-week-old female athymic mice for 14 days. The results demonstrated that the hADSCs could be efficiently induced into urothelial lineage by indirect coculture. The differentiated cells seeded onto the PLA/collagen ureteral scaffolds survived up to 7 days and maintained proliferation in vitro, which indicated that the scaffolds displayed good biocompatibility. In vivo study showed that the differentiated cells in the grafts survived, formed multiple layers on the scaffolds and expressed urothelial lineage markers. In conclusion, hADSCs may serve as an alternative cell resource in cell-based tissue engineering for ureteral reconstruction. These cells could be employed to construct a model of ureteral engineering grafts and be effectively applied in vivo, which could be a new strategy on ureteral replacement with applicable potential in clinical research. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:2612-2622, 2012.     

Increased apoptosis in gastric mucosa adjacent to intestinal metaplasia

BACKGROUND: The biological processes involved in the development of gastric mucosal atrophy and intestinal metaplasia are still incompletely understood. Reports testing the hypothesis that apoptosis leads to atrophy have yielded conflicting results. The availability of new antibodies for the detection of apoptotic cells in tissue sections has facilitated the analysis of the role of apoptosis in the gastritis-atrophy-intestinal metaplasia sequence. METHODS: Archival material from 40 gastric resection specimens with normal mucosa (n = 5), chronic active gastritis (n = 17), or intestinal metaplasia (n = 18) was studied. Immunohistochemistry was performed using antibodies directed against cleaved cytokeratin 18 and active caspase 3. Slides were scored on a 0-3 scale for the presence of apoptotic cells. RESULTS: Normal gastric mucosa contained low numbers of apoptotic cells at the surface epithelium (mean score, 0.20). This number was significantly increased in cases with chronic gastritis (mean score, 1.06) and in those with intestinal metaplasia (mean score, 2.56). Within the intestinal metaplasia cases, 44 different foci of intestinal metaplasia were identified. In 39 of these 44 areas, concentrations of apoptotic cells were seen immediately adjacent to the foci of intestinal metaplasia, but not in the metaplastic epithelium itself. CONCLUSIONS: Apoptosis is uncommon in normal gastric mucosa. Chronic inflammation and intestinal metaplasia are associated with increased apoptosis, but occur mainly at the mucosal surface and not in the deeper layers. These findings do not support the concept that apoptosis underlies the loss of gastric glands and leads to atrophy, but the observed concentration of apoptotic epithelial cells adjacent to foci of intestinal metaplasia could be related to heterogeneity of epithelial damage, causing apoptosis, to which intestinal metaplasia is a response.     

Bone marrow genesis after subcutaneous delivery of rat osteogenic cell-seeded biodegradable scaffolds into nude mice

This study describes the generation of an active hematopoietic marrow within the confines of a biodegradable, macroporous polyester scaffold, seeded with rat osteogenic cells, after subcutaneous implantation in nude mice. A macroporous, poly(DL-lactide-co-glycolide) polymer scaffold, into which resorbable calcium phosphate particles were incorporated, was seeded with rat bone marrow-derived cells. Scanning electron microscopy of the cell-seeded scaffold demonstrated confluent cell colonization. Scaffolds seeded with cells were implanted under the dorsum of immunocompromised mice for 5 weeks. Histological analysis revealed bone formation along the scaffold pores creating bony cavities within which a host-derived, hematopoietic marrow was observed which included hematopoietic precursors, megakaryocytes, fat cells, and numerous marrow sinusoids. In those areas where bone was not elaborated on the scaffold surface, no marrow genesis was observed and the scaffold interstices were filled with fibrous tissue. These results demonstrate the utility of this biodegradable scaffold in delivery of a phenotypically functional cell population for bone tissue and bone marrow engineering applications. Moreover, the recapitulation of hematopoietic marrow tissue within the engineered bony cavities also provides a new experimental environment with which to further investigate the interactions of hematopoietic and nonhematopoietic compartments of the marrow microenvironment.     

Structural reactions of the buccal mucosa in diabetic parodontopathy

Pronounced changes in the capillaries, hemodynamic disorders, epitheliocyte degeneration and atrophy develop in the buccal mucosa of patients with types I and II diabetes mellitus in the absence of inflammatory cellular infiltration or with facultative infiltration. The morphogenesis of pathological changes can be regarded as primary diabetic microangiopathy causing metabolic disorders with the development of degenerative and atrophic changes in all structural components of the buccal mucosa and development of diabetic parodontopathy, a primary degenerative process. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 581–584, November, 2006     

Mucin profiles in the mucosa adjacent to large bowel non-adenocarcinoma neoplasias

Morphological features and mucin secretion patterns were investigated in the colonic mucosa adjacent to or overlying mesenchymal or primary epithelial neoplasias, other than adenomas and adenocarcinomas. The material included 15 cases of non-adenocarcinoma tumours examined during 1978-1981. Increased sialomucins and morphological features similar to those described in the so-called 'transitional' mucosa adjacent to primary colorectal adenocarcinomas were observed in only two cases. In contrast our previous studies have demonstrated 'transitional' profiles in 98% of adenocarcinomas.     

The effect of long-term release of Shh from implanted biodegradable microspheres on recovery from spinal cord injury in mice

After spinal cord injury (SCI), loss of cells and damage to ascending and descending tracts can result in paralysis. Current treatments for SCI are based on patient stabilization, and much-needed regenerative therapies are still under development. To activate and instruct stem and progenitor cells or injured tissue to aid SCI repair, it is important to modify the injury environment for a protracted period, to allow time for cell activation, proliferation and appropriate fate differentiation. Shh plays a critical role in spinal cord formation, being involved in multiple processes: it promotes production of motor neurons and oligodendrocytes from ventral cord progenitor cells and serves as an axon guidance molecule. Hence Shh is a candidate pleiotropic beneficial environmental factor for spinal cord regeneration. Here we show that administration of biodegradable microspheres that provide sustained, controlled delivery of Shh resulted in significant functional improvement in two different mouse models of SCI: contusion and dorsal hemioversection. The mechanism is multifactorial, involving increased proliferation of endogenous NG2+ oligodendrocyte lineage cells, decreased astrocytic scar formation and increased sprouting and growth of corticospinal (CST) and raphespinal tract (RST) fibers. Thus, long-term administration of Shh is a potential valuable therapeutic intervention for SCI.     

Use of stereolithography to manufacture critical-sized 3D biodegradable scaffolds for bone ingrowth

A novel approach to the manufacture of biodegradable polymeric scaffolds for tissue-engineering utilizing stereolithography (SLA) is presented. SLA is a three-dimensional (3D) printing method that uses an ultraviolet laser to photo-crosslink a liquid polymer substrate. The current generation of SLA devices provide a 3D printing resolution of 0.1 mm. The experiments utilized a biodegradable resin mixture of diethyl fumarate (DEF), poly(propylene fumarate) (PPF), and a photoinitiator, bisacylphosphine oxide (BAPO). The PPF is crosslinked with the use of the SLA's UV laser (325-nm wavelength). An SLA device was retrofitted with a custom fixture build tank enclosing an elevator-driven build table. A 3D prototype model testing the manufacturing control this device provides was created in a computer-aided-design package. The resulting geometric data were used to drive the SLA process, and a DEF/PPF prototype part was successfully manufactured. These scaffolds have application in the tissue engineering of bony substrates.     

Potential antiosteoporosis effect of biodegradable magnesium implanted in STZ-induced diabetic rats

Pure magnesium (Mg) was implanted intramedullary into the femur of streptozotocin (STZ)-induced diabetic rats to investigate its effect on bone growth after 6 weeks degradation. The experimental results showed that the femoral BMD in diabetic rats was significantly lower than that in controls (p < 0.01) but restored notably by Mg implantation. The contents of calcium (Ca), phosphorus (P), Mg, zinc (Zn), potassium (K), strontium (Sr), and sulfur (S) in bone of diabetic group were significantly lower than those in controls but remarkably increased with implantation of Mg. The residual weight calculation showed that 29.41% of Mg was degraded in vivo. The energy dispersive X-ray spectroscopy (EDS) analysis showed that the reaction layer on the surface of the Mg implant mainly consisted of C, Ca, O, P, and Mg. Besides, serum Mg level was significantly decreased in diabetic group compared with the control group but increased by Mg treatment. Also, there were no significant differences in body weight and blood glucose, as well as blood glycosylated hemoglobin (HbAIc%), serum Ca, alanine aminitransperase (ALT), aspartate aminotransferase (AST), uric acid (UA), nonesterified fatty acid (NEFA), cholinesterase (CHE), and creatinine (CR) levels between diabetic and Mg-implanted rats. The study indicated that Mg implant had no obvious toxicity in STZ-induced diabetic rats and may act as a potential agent to treat osteoporosis.     

Tissue reaction to matrices of reconstituted keratin polymer implanted subcutaneously in sheep

Four different modifications of the keratin polymer were made as small rods and inserted into the subcutaneous tissue of the hind limbs of adult female sheep. Material 1 was porous, whereas materials 2, 3, and 4 had little or no internal porosity. The tissue response to each material was determined with regard to the extent of the inflammatory reaction and formation of a fibrous capsule around the implant, and the integrity and morphological appearance of the implant was assessed. An inflammatory cell infiltrate and fibrous capsule formed at an early stage around implants of material 1. Subsequently the inflammation decreased, the fibrous capsule became more mature, and the implants became cavitated and invaded by mono- and multi-nucleated macrophages, fibroblasts and blood vessels, with breakdown of the implant material occurring at its periphery. Similar changes were observed for implants made of materials 2 and 4. Implants of material 3 were remarkable in that, while surrounded by an inflammatory infiltrate and a fibrous capsule, they did not show any disturbance even at 24 weeks. The fibrous capsule around material 3 was thinner than that around material 1 at 6 to 24 weeks (both materials prepared using ammonium thioglycollate). No difference in capsule thickness was found for materials 2 and 4 (both materials prepared using thioglycollic acid).     

Hyperplastic polyps of the gastric mucosa adjacent to gastroenterostomy stomas.

In two patients with functionally active, nonatrophic oxyntic gastric mucosa, hyperplastic polyps developed on the gastric side of Billroth II and Billroth I anastomoses. The polyp associated with the Billroth II procedure was almost circumferential, sparing only the lesser curvature. That associated with the Billroth I operation was on the anterior wall from lesser to greater curvature. The operations had been performed 11 years and ten years earlier for peptic ulcers of the gastroduodenal junction. The sites of polyp formation resembled those of experimental cancers in rats with Billroth I and Billroth II anastomoses, and of human cancer occurring 20 or more years after gastrojejunostomy for benign disease. This suggests that stomal carcinomas and hyperplastic polyps each result from the reflux of enteric contents into the stomach remnant.     

Failure to demonstrate specificity of the morphological and histochemical changes in mucosa adjacent to colonic carcinoma (transitional mucosa).

Characteristic morphological and histochemical changes in mucosa adjacent to colorectal adenocarcinomas have been described. It has been suggested that this type of mucosa, labelled transitional mucosa (TM) because of its specific association with colorectal adenocarcinoma, is indicative of a premalignant change. In an investigation of mucosa adjacent to anal tumours extending into the rectum and mucosa from solitary ulcer syndrome and colostomies the mucosal alterations described in TM could be demonstrated. Thus TM is not specifically related to colorectal adenocarcinoma and probably arises as a secondary phenomenon. The claim that TM represents a premalignant change cannot be substantiated.     

Induction of angiogenesis by hyperplastic colonic mucosa adjacent to colon cancer

We determined whether hyperplastic mucosa adjacent to colon cancer contributes to neoplastic angiogenesis. Surgical specimens of human colon cancer (40 Dukes' stage B and 34 Dukes' stage C) were analyzed by immunohistochemistry for expression of proliferative and angiogenic molecules. The mucosa adjacent to Dukes' stage C tumors (but not Dukes' stage B tumors) had a higher Ki-67 labeling index and a higher expression of epidermal growth factor receptor and transforming growth factor-alpha than distant mucosa. The expression levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8, and the vascular density in the adjacent mucosa were similar to those in the tumor lesions and significantly higher than those in the distant mucosa. The expression of interferon-beta inversely correlated with the level of pro-angiogenic molecules and the vascular density. The injection of metastatic human colon cancer cells and murine colon cancer cells into the cecal wall of mice induced hyperplastic changes in the adjacent mucosa which expressed higher levels of epidermal growth factor receptor, basic fibroblast growth factor, and vascular endothelial growth factor, and lower levels of interferon-beta than did the control mucosa, which directly correlated with the degree of hyperplasia. These data suggest that metastatic human colon cancer cells can induce hyperplasia in the adjacent mucosa, which in turn produces angiogenic molecules that contribute to neoplastic angiogenesis.     

Tissue ingrowth and degradation of two biodegradable porous polymers with different porosities and pore sizes

Commonly, spontaneous repair of lesions in the avascular zone of the knee meniscus does not occur. By implanting a porous polymer scaffold in a knee meniscus defect, the lesion is connected with the abundantly vascularized knee capsule and healing can be realized. Ingrowth of fibrovascular tissue and thus healing capacity depended on porosity, pore sizes and compression modulus of the implant. To study the lesion healing potential, two series of porous polyurethanes based on 50/50 epsilon-caprolactone/L-lactide with different porosities and pore sizes were implanted subcutaneously in rats. Also, in vitro degradation of the polymer was evaluated. The porous polymers with the higher porosity, more interconnected macropores, and interconnecting micropores of at least 30 microm showed complete ingrowth of tissue before degradation had started. In implants with the lower macro-porosity and micropores of 10-15 microm degradation of the polymer occurred before ingrowth was completed. Directly after implantation and later during degradation of the polymer, PMN cells infiltrated the implant. In between these phases the foreign body reaction remained restricted to macrophages and giant cells. We can conclude that both foams seemed not suited for implantation in meniscal reconstruction while either full ingrowth of tissue was not realized before polymer degradation started or the compression modulus was too low. Therefore, foams must be developed with a higher compression modulus and more connections with sufficient diameter between the macropores.