Histomorphological Properties Research Articles

Autologous Cryopreserved Adipose Tissue Using an Innovative Technique: An In Vitro Biological Characterization

Abstract Background Utilization of autologous adipose tissue transplantation in plastic and orthopedic surgery such as breast reconstruction and intra-articular injection has become an attractive surgical treatment with satisfactory clinical outcomes. Nevertheless, repeated liposuctions necessary to harvest fatty tissue, normally performed with sedation or general anesthesia, may represent a noteworthy concern. Objectives The aim of this study was to demonstrate through an in vitro characterization the validity of the surgical option of cryopreserved autologous adipose tissue harvested in a single shot for repeated graft transfer in breast reconstruction without impairment of cell viability and sterility. Methods Adipose tissue was collected by standard liposuction from patients who needed numerous fat grafting procedures for breast reconstruction. According to an innovative and patented cryopreservation method, autologous adipose tissue was subsequently fractioned in a sterile bag system and frozen at the RER Tissue Bank of the Emilia Romagna Region. Each graft was evaluated for sterility and cell viability immediately after harvesting, and 1, 3, 6, 12, and preliminarily 18 months after cryopreservation and thawing. Results In vitro results showed that after processing, middle-term and long-term cryopreservation, and subsequent thawing, autologous cryopreserved adipose tissue retained absence of bacterial contamination, high cellular viability, and unmodified histomorphological properties, thereby ensuring maintenance of the stromal vascular niche and the filling properties in different multistep surgical procedures. Conclusions In vitro study and sterility assessment showed that autologous cryopreserved adipose tissue grafting is a safe procedure, making it possible to avoid multiple liposuction surgery. No impairment of sterility, cell viability, or morphology was observed over time.

Repeated birth injuries lead to long-term pelvic floor muscle dysfunction in the preclinical rat model

ObjectivesVaginal childbirth is a key risk factor for pelvic floor muscle injury and dysfunction, and subsequent pelvic floor disorders. Multiparity further exacerbates these risks. Using the rat model, validated for the studies of the human pelvic floor muscles, we have previously identified that a single simulated birth injury results in pelvic floor muscle atrophy and fibrosis. We hypothesized that multiple birth injuries would further overwhelm the muscle regenerative capacity, leading to functionally relevant pathological alterations long-term. Study DesignSprague-Dawley rats underwent simulated birth injury and were allowed to recover for 8 weeks before undergoing additional birth injury. Animals were sacrificed at acute (3- and 7-days post injury), subacute (21-, 28-, and 35-days post-injury), and long-term (8- and 12-weeks post-injury) time points post-second injury (N=3-8/time point), and the pubocaudalis portion of the rat levator ani complex was harvested to assess the impact of repeated birth injuries on muscle mechanical and histomorphological properties. The accompanying transcriptional changes were assessed by a customized NanoString panel. Uninjured animals were used as controls. Data with a parametric distribution were analyzed by a two-way analysis of variance followed by post hoc pairwise comparisons using Tukey’s or Sidak’s tests; non-parametrically distributed data were compared with Kruskal-Wallis test followed by pairwise comparisons with Dunn’s test. Data, analyzed using GraphPad Prism v8.0, San Diego, CA, are presented as mean ± SEM or median (range). ResultsFollowing the 1st simulated birth injury, active muscle force decreased acutely relative to uninjured controls (12.9±0.9 vs 25.98±2.1 g/mm2, P<0.01). At 4 weeks, muscle active force production recovered to baseline and remained unchanged at 8 weeks after birth injury (P>0.99). Similarly, precipitous decrease in active force was observed immediately after repeated birth injury (18.07±1.2 vs 25.98±2.1 g/mm2, P<0.05). In contrast to the functional recovery after a single birth injury, a long-term decrease in muscle contractile function was observed up to 12 weeks after repeated birth injuries (18.3±1.6 vs 25.98±2.1 g/mm2, P<0.05). Fiber size was smaller at the long-term time points after 2nd injury compared to the uninjured group (12 weeks vs uninjured control: 1485 (60.7-5000) vs 1989 (65.6-4702) μm2, P<0.0001). The proportion of fibers with centralized nuclei, indicating active myofiber regeneration, returned to baseline at 8 weeks post-1st birth injury, (P=0.95), but remained elevated as far as 12 weeks post-2nd injury (12 weeks vs uninjured control: 7.1±1.5 vs 0.84±0.13%, P<0.0001). In contrast to the plateauing intramuscular collagen content after 4 weeks post-1st injury, fibrotic degeneration increased progressively over 12 weeks after repeated injury (12 weeks vs uninjured control: 6. 7±1.1 vs 2.03±0.2%, P<0.001). Prolonged expression of pro-inflammatory genes accompanied by a greater immune infiltrate was observed after repeated compared to a single birth injury. ConclusionsOverall, repeated birth injuries led to a greater magnitude of pathological alterations compared to a single injury, resulting in more pronounced pelvic floor muscle degeneration and muscle dysfunction in the rat model. The above provides a putative mechanistic link between multiparity and the increased risk of pelvic floor dysfunction in women.

Immunomodulatory extracellular matrix hydrogel induces tissue regeneration in a model of partial glossectomy

While oropharyngeal cancer treatment regimens, including surgical resection, irradiation, and chemotherapy, are effective at removing tumors, they lead to muscle atrophy, denervation, and fibrosis, contributing to the pathogenesis of oropharyngeal dysphagia – difficulty swallowing. Current standard of care of rehabilitative tongue strengthening and swallowing exercises is ineffective. Here, we evaluate an alternative approach utilizing an acellular and injectable biomaterial to preserve muscle content and reduce fibrosis of the tongue after injury. Skeletal muscle extracellular matrix (SKM) hydrogel is fabricated from decellularized porcine skeletal muscle tissue. A partial glossectomy injury in the rat is used to induce tongue fibrosis, and SKM hydrogels along with saline controls are injected into the site of scarring two weeks after injury. Tissues are harvested at 3 and 7 days post-injection for gene expression and immunohistochemical analyses, and at 4 weeks post-injection to evaluate histomorphological properties. SKM hydrogel reduces scar formation and improves muscle regeneration at the site of injury compared to saline. SKM additionally modulates the immune response towards an anti-inflammatory phenotype. This study demonstrates the immunomodulatory and tissue-regenerative capacity of an acellular and minimally invasive ECM hydrogel in a rodent model of tongue injury.

Efficacy of Flowable Collagen Hemostat Evaluated in Preclinical Models of Liver Injury and Spinal Cord Exposure.

Excessive bleeding in trauma and surgical settings leads to increased operative time, reoperation rates, and overall healthcare costs. A wide range of hemostatic agents have been developed to control bleeding that can vary considerably in type of hemostatic action, ease of application, cost, risk of infection, and dependence on patient coagulation. Microfibrillar collagen-based hemostatic materials (MCH) have yielded beneficial results in a variety of applications. A new flowable collagen product, containing a modified MCH flour, but in a more convenient flowable delivery system, was evaluated for hemostatic efficacy in preclinical models of solid organ injury and spinal cord exposure. The primary objective of this study was to compare the hemostatic potential and local tissue responses to this novel, flowable collagen-based hemostatic agent to the original flour formulation to confirm that the new method of delivery did not interfere with the hemostatic properties of the MCH flour. When observed visually, the flowable MCH flour mixed with saline (FL) provided more precise application and uniform coverage to injured tissues compared to the dry MCH flour alone (F0). All of the treatments (FL, F0, and gauze) exhibited comparable Lewis bleed grade at all three time points evaluated in the capsular resection liver injury model (bleed grade: 1.0-1.3; p> 0.05 in all cases). FL and F0 exhibited comparable 100% acute hemostatic efficacy and similar long-term histomorphological properties (up to 120 days) in a capsular resection liver injury in pigs, while gauze resulted in significantly lower rates of acute hemostatic efficacy (8-42%, p<0.05 in all cases). In an ovine model of dorsal laminectomy and durotomy, FL and F0 again exhibited comparable results without any neurological effects. Flowable microfibrillar collagen was shown to yield favorable short- and long-term outcomes in two representative applications where hemostatic efficacy is critical to surgical success.

Relationship between computed tomography and histological features of gastrointestinal stromal tumors

Background and Aim: Histomorphological and immunohistochemical (IHC) properties of gastrointestinal stromal tumors (GISTs) allow for accurate diagnosis and determine the prognosis. We aimed to evaluate the relationship between the computed tomography (CT) features, histomorphological properties, and IHC markers. Materials and Methods: This retrospective study comprised patients with pathologically confirmed GISTs between 2016 and 2021. The predefined CT characteristics comprised tumor size, hemorrhage and calcification, CT-growth pattern (exophytic/endophytic), and contrast enhancement pattern of the solid component (homogeneous/heterogeneous). The GISTs were divided into groups according to the National Institutes of Health risk category, cell type, presence of necrosis, CD117 and α-SMA positivity, and Ki-67 index. The frequencies of CT phenotypes were compared between groups. Results: Overall 24 (14 [58.3%] males) patients with a median age of 64 (59.5–75.5) having 25 GISTs were included. Of 25 GISTs, 16 (64%) were gastric and 9 (36%) were intestinal in origin. Among CT features, the maximum diameter was higher in epithelioid, infiltrative, a mitotic count ≥5/50, necrotic, high-risk GISTs (P &lt; 0.05 for all). The median tumor size was higher in Ki-67 &gt;8 than Ki-67 &lt;6 subjects (112.5 [39.25–153.75] vs. 22.5 [16.75–57.5] mm, P = 0.014). A heterogeneous enhancement was also more frequent in Ki-67 &gt;8 tumors (P = 0.04). The enhancement pattern did not differ according to CD-117 or SMA positivity. Logistic regression analysis revealed that the only independent predictor of a Ki-67 &gt;8 status was the tumor size (odds ratio: 1.02, 95% confidence interval: 1.001–1.046, P = 0.04). Conclusions: Heterogeneously enhanced large GISTs at CT imaging strongly suggest the presence of poor prognostic factors including a high Ki-67 index and/or high-risk category.

Salivary Gland Adaptation to Dietary Inclusion of Hydrolysable Tannins in Boars.

Simple SummaryTannins have traditionally been avoided in animal nutrition due to their anti-nutritive effects. However, recent studies reported hydrolysable tannins as beneficial additives that have antimutagenic, anticarcinogenic, antidiarrheal, and antiulcerogenic effects on animals. In a study testing the inclusion of hydrolysable tannins as a potential nutritive factor to reduce boar taint in entire males, significant enlargement of the parotid glands (parotidomegaly) was observed. In this study, we aimed to determine the morphological and immunohistochemical basis for the observed parotidomegaly. We discovered that enlargement of glandular lobules and acinar area, an increased ratio between the nucleus and cytoplasm of serous cells, and increased excretion of proline-rich proteins (PRPs) were characteristic of the experimental group that received the highest dietary tannin intake. The mandibular salivary gland, on the other hand, did not show significant morphological changes among the experimental groups. This suggests increased functional activity of the parotid salivary glands as the first and most important line of defense against high dietary tannin and its potential negative effects.The ingestion of hydrolysable tannins as a potential nutrient to reduce boar odor in entire males results in the significant enlargement of parotid glands (parotidomegaly). The objective of this study was to characterize the effects of different levels of hydrolysable tannins in the diet of fattening boars (n = 24) on salivary gland morphology and proline-rich protein (PRP) expression at the histological level. Four treatment groups of pigs (n = 6 per group) were fed either a control (T0) or experimental diet, where the T0 diet was supplemented with 1% (T1), 2% (T2), or 3% (T3) of the hydrolysable tannin-rich extract Farmatan®. After slaughter, the parotid and mandibular glands of the experimental pigs were harvested and dissected for staining using Goldner’s Trichrome method, and immunohistochemical studies with antibodies against PRPs. Morphometric analysis was performed on microtome sections of both salivary glands, to measure the acinar area, the lobular area, the area of the secretory ductal cells, and the sizes of glandular cells and their nuclei. Histological assessment revealed that significant parotidomegaly was only present in the T3 group, based on the presence of larger glandular lobules, acinar areas, and their higher nucleus to cytoplasm ratio. The immunohistochemical method, supported by color intensity measurements, indicated significant increases in basic PRPs (PRB2) in the T3 and acidic PRPs (PRH1/2) in the T1 groups. Tannin supplementation did not affect the histo-morphological properties of the mandibular gland. This study confirms that pigs can adapt to a tannin-rich diet by making structural changes in their parotid salivary gland, indicating its higher functional activity.

Relationships between broiler final weights and histomorphometric parameters of certain segments of the intestine

The aim of this study was to determine relationships between final mass of broilers and the histomorphological properties of individual segments of the gastrointestinal tract. This is confirmed by the obtained results, which indicate a strong and significant correlation between compared parameters. A strong significant (p<0.05) correlation (r=0.866) was found between the broiler final weight and the duodenal villus length, and between the broiler final weight and the caecal villus length (r=0.918). Correlation between the broiler final weight and the duodenal villus width (r=0.841), as well the caecal villi width (r=0.918) was strongly significant (p<0.05). Between the crypt depths in caecum correlation was medium and significant (r=0.701, p<0.05). It was determined that between the broiler final weights and the ratio of villus length and crypt depth there is a significant medium correlation (r = 0.736, p<0.05). A strong (r = 0.924) significant (p <0.05) correlation was found between the broiler final weight and the ratio of villus length and caecal crypt depth. Between final mass of broilers and other histomorphological properties of individual segments of intestine there was no significant correlation.

Retraction Note to: Mesenchymal stem cells seeded onto tissue-engineered osteoinductive scaffolds enhance the healing process of critical-sized radial bone defects in rat.

Long bone defects comprise one of the most prevalent clinical problems worldwide and the current bone grafting materials have major limitations to repair them. Although tremendous efforts have been made to repair critical-sized long bone defects in animal models, designing an optimal bone tissue-engineered substitute remains one of the main challenges. Hence, this study aims to closely mimic a natural bone healing process by a tissue-engineered construct including osteoinductive materials pre-seeded with bone marrow-derived mesenchymal stem cells (BMSCs). Bioactive glass (BG) was incorporated into the gelatin/nano-hydroxyapatite (G/nHAp) scaffold (conventional one) to improve the bone regeneration process via its osteoinductivity and angiogenic activity. The fabricated G/nHAp and gelatin/nano-hydroxyapatite/bioactive glass (G/nHAp/BG) scaffolds were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) and analyzed for porosity and degradation rate. The osteogenic capability of fabricated scaffolds with or without BMSCs was then evaluated in vitro and in vivo. Critical-sized radial bone defects in rats were randomly filled with cell-free and BMSC-seeded scaffolds, autograft and a group left empty without any treatment. In vitro analysis showed that the G/nHAp/BG scaffold significantly increased the expression level of osteogenic and angiogenic markers in comparison to the G/nHAp-treated and control groups (P < 0.05). Moreover, the defects treated with the BMSC-seeded scaffolds showed superior bone formation and structural properties compared to the cell-free scaffolds 4 and 12 weeks post surgery. The radiological and histomorphological properties of defects treated by BMSC-seeded scaffolds, especially the BMSC-seeded G/nHAp/BG scaffold, were comparable to those of the autograft group. It is concluded that the combination of osteoconductive materials (i.e., nHAp) with the bioactive ones such as bioactive glass can effectively accelerate the bone regeneration process. In addition, our results demonstrated that the BMSCs have the potential to drastically increase the bone regeneration ability of osteoinductive scaffolds.

Low-speed drilling without irrigation versus conventional drilling for dental implant osteotomy preparation: a systematic review.

This paper aims to review the evidence comparing low-speed drilling without irrigation versus conventional drilling for dental implant osteotomy preparation. A systematic review was carried out based on the PRISMA statement. Four databases and gray literature were searched up to November 2020. In vitro, animal, and clinical studies were included. The variables were temperature change, drilling time, quantity of harvested bone, osteotomy precision, marginal bone loss, implant success rate, osseointegration, and the histomorphologic characteristics and cellularity of the osteotomy and of the harvested bone. Different tools for the assessment of bias were applied for each study design. A total of 626 articles were identified, of which 13 were included. Both low-speed drilling without irrigation (test group) and conventional drilling (control group) maintained temperatures below the critical temperature of 47 °C. The test group yielded a greater quantity and more beneficial cellular and histomorphologic properties of harvested bone, with a longer drilling time and greater osteotomy precision (p < 0.05). No significant results were obtained regarding drill wear, osseointegration, marginal bone loss, implant success rate, and histomorphology of the dental implant osteotomy (p > 0.05). The results, in particular on the osteotomy precision and quantity of harvested bone chips, should be interpreted with caution because outcomes are based in only one in vitro study. Low-speed drilling without irrigation seems to be comparable to conventional drilling in preparing dental implant osteotomies. In some situations, low-speed drilling without irrigation might offer advantages over conventional drilling. The results should be interpreted with caution due to the low percentage of clinical human studies. Accordingly, more clinical studies are needed to improve the scientific evidence on this topic. The low-speed drilling without irrigation is a valid technique for dental implant osteotomy preparation. Its higher quantity and quality of harvested autologous bone might be particularly beneficial in cases of dental implant placement with minor simultaneous bone regeneration.

Artificial Intelligence in Pathology.

Increasing digitalization enables the use of artificial intelligence (AI) and machine learning in pathology. However, these technologies have only just begun to be implemented, and no randomized prospective trials have yet shown a benefit of AI-based diagnosis. In this review, we present current concepts, illustrate them with examples from representative publications, and discuss the possibilities and limitations of their use. This article is based on the results of a search in PubMed for articles published between January 1950 and January 2020 containing the searching terms "artificial intelligence," "deep learning," and "digital pathology," as well as the authors' own research findings. Current research on AI in pathology focuses on supporting routine diagnosis and on prognostication, particularly for patients with cancer. Initial data indicate that pathologists can arrive at a diagnosis faster and more accurately with the aid of a computer. In a pilot study on the diagnosis of breast cancer, involving 70 patients, sensitivity for the detection of micrometastases rose from 83.3% (by a pathologist alone) to 91.2% (by a pathologist combined with a computer algorithm). The evidence likewise suggests that AI applied to histomorphological properties of cells during microscopy may enable the inference of certain genetic properties, such as mutations in key genes and deoxyribonucleic acid (DNA) methylation profiles. Initial proof-of-concept studies for AI in pathology are now available. Randomized, prospective studies are now needed so that these early findings can be confirmed or falsified.

The macroscopic and histomorphological properties of periosteal rib lesions and its relation with disease duration: evidence from the Luis Lopes Skeletal Collection (Lisbon, Portugal).

Periosteal new bone formation (PNBF) is a common finding in a large spectrum of diseases. In clinical practice, the morphology and location of periosteal lesions are frequently used to assist in the differential diagnosis of distinct bone conditions. Less commonly reported is the presence of PNBF on the ribs. This contrasts with the data retrieved from the study of skeletonized human remains that shows a high frequency of cases and a strong, albeit not specific, association between periosteal rib lesions and pulmonary conditions (e.g. tuberculosis). Despite that, an overall disagreement regarding the specificity and non-specificity of periosteal reactions exists in the study of dry bone remains. The insufficient number of clinical models exploring the morphology and the pathophysiology of PNBF's and the lack of systematic studies of pathological samples with a known diagnosis are claimed as major reasons for the disagreements. This study aimed to describe and compare the macroscopic and the histomorphologic appearance of periosteal rib lesions and to discuss their usefulness as diagnostic indicators. To pursue this goal, an assemblage of 13 rib samples (males=11, females=2, mean age-at-death=36.6years old) was collected from the Luis Lopes Skeletal Collection (Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Portugal). The assemblage belongs to individuals who died from pulmonary-TB (group 1), non-TB pulmonary infections (group 2) and other conditions (group 3). Prior to sample preparation, the ribs were visually inspected and the PNBF described according to its thickness, the degree of cortical integration and the type of new bone formed (e.g. woven, lamellar or both). After sampling, each bone sample was prepared for histological analysis under plane and polarized light microscopy. Macroscopically, the results showed no differences in the new bone composition between cause-of-death groups. Only slight differences in the degree of cortical integration, which was most frequently classified as mild to high in the pulmonary-TB group, were observed. Histologically, no distinguishing features were identified by pathological group. However, new bone microarchitectures were observed compatible with (1) acute, fast-growing processes (e.g. spiculated reactions), (2) long-standing processes with a rapid bone formation (e.g. appositional layering of bone) and/or (3) chronic, slow-growing processes (e.g. layers of compact lamellae). To some extent, these distinct rates of disease progression resonate with the cause-of-death listed for some individuals. Despite the small sample size, the results of this investigation are in agreement with previous studies, according to which the macroscopic and histological appearance of periosteal formations are not specific for a particular pathological conditions. Nevertheless, the results support the conclusion that the morphology of periosteal lesions is a good biological indicator for inferring the rate of progression and duration of pathological processes. This study provides important reference data regarding the histomorphology of periosteal lesions that can be used for comparative purposes, as well as to narrow down the differential diagnosis in unidentified skeletal remains.

The role of subchondral bone, and its histomorphology, on the dynamic viscoelasticity of cartilage, bone and osteochondral cores

SummaryObjectiveViscoelastic properties of articular cartilage have been characterised at physiological frequencies. However, studies investigating the interaction between cartilage and subchondral bone and the influence of underlying bone histomorphometry on the viscoelasticity of cartilage are lacking.MethodDynamic Mechanical Analysis (DMA) has been used to quantify the dynamic viscoelasticity of bovine tibial plateau osteochondral cores, over a frequency sweep from 1 to 88 Hz. Specimens (approximately aged between 18 and 30 months) were neither osteoarthritic nor otherwise compromised. A maximum nominal stress of 1.7 MPa was induced. Viscoelastic properties of cores have been compared with that of its components (cartilage and bone) in terms of the elastic and viscous components of both structural stiffness and material modulus. Micro-computed tomography scans were used to quantify the histomorphological properties of the subchondral bone.ResultsOpposing frequency-dependent loss stiffness, and modulus, trends were witnessed for osteochondral tissues: for cartilage it increased logarithmically (P < 0.05); for bone it decreased logarithmically (P < 0.05). The storage stiffness of osteochondral cores was logarithmically frequency-dependent (P < 0.05), however, the loss stiffness was typically frequency-independent (P > 0.05). A linear relationship between the subchondral bone plate (SBP) thickness and cartilage thickness (P < 0.001) was identified. Cartilage loss modulus was linearly correlated to bone mineral density (BMD) (P < 0.05) and bone volume (P < 0.05).ConclusionThe relationship between the subchondral bone histomorphometry and cartilage viscoelasticity (namely loss modulus) and thickness, have implications for the initiation and progression of osteoarthritis (OA) through an altered ability of cartilage to dissipate energy.

Evaluation of dexamethasone treated mesenchymal stem cells for recovery in neurotmesis model of peripheral nerve injury

ABSTRACTObjective: Peripheral nerve injuries comprise significant portion of the nervous system injuries. Although peripheral nerves show some capacity of regeneration after injury, the extent of regeneration is not remarkable. The present study aimes to evaluate the regeneration of transected sciatic nerve by a therapeutic value of dexamethasone (DEX) associated with cell therapy (Cell) and biodegradable membrane (Mem) in rat.Methods: Male Wistar rats (n = 42, 180-200g) were randomly divided into control (Ctrl), Membrane+ Cell, Mem, DEX, DEX+ Cell, DEX+ Mem and DEX+ Cell+ Mem groups. Functional recovery was evaluated at 2, 4, 6, 8 and 12 weeks after surgery using sciatic functional index (SFI), withdrawal reflex latency (WRL) test, electrophysiological and histological analyses.Results: The rats in the DEX+ Cell+ Mem-treated group showed a significant improvement in SFI, WRL and electrophysiological findings during the 2nd to 12th weeks after surgery. In addition, histomorphological findings showed a significant improvement in the DEX+ Cell+ Memtreated group, at 12 weeks after surgery.Discussion: Taken together, use of DEX associated with cell and biodegradable membrane could improve functional and histomorphological properties of the sciatic nerve after injury.

Mesenchymal stem cells seeded onto tissue-engineered osteoinductive scaffolds enhance the healing process of critical-sized radial bone defects in rat.

Long bone defects comprise one of the most prevalent clinical problems worldwide and the current bone grafting materials have major limitations to repair them. Although tremendous efforts have been made to repair critical-sized long bone defects in animal models, designing an optimal bone tissue-engineered substitute remains one of the main challenges. Hence, this study aims to closely mimic a natural bone healing process by a tissue-engineered construct including osteoinductive materials pre-seeded with bone marrow-derived mesenchymal stem cells (BMSCs). Bioactive glass (BG) was incorporated into the gelatin/nano-hydroxyapatite (G/nHAp) scaffold (conventional one) to improve the bone regeneration process via its osteoinductivity and angiogenic activity. The fabricated G/nHAp and gelatin/nano-hydroxyapatite/bioactive glass (G/nHAp/BG) scaffolds were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) and analyzed for porosity and degradation rate. The osteogenic capability of fabricated scaffolds with or without BMSCs was then evaluated in vitro and in vivo. Critical-sized radial bone defects in rats were randomly filled with cell-free and BMSC-seeded scaffolds, autograft and a group left empty without any treatment. In vitro analysis showed that the G/nHAp/BG scaffold significantly increased the expression level of osteogenic and angiogenic markers in comparison to the G/nHAp-treated and control groups (P < 0.05). Moreover, the defects treated with the BMSC-seeded scaffolds showed superior bone formation and structural properties compared to the cell-free scaffolds 4 and 12weeks post surgery. The radiological and histomorphological properties of defects treated by BMSC-seeded scaffolds, especially the BMSC-seeded G/nHAp/BG scaffold, were comparable to those of the autograft group. It is concluded that the combination of osteoconductive materials (i.e., nHAp) with the bioactive ones such as bioactive glass can effectively accelerate the bone regeneration process. In addition, our results demonstrated that the BMSCs have the potential to drastically increase the bone regeneration ability of osteoinductive scaffolds.

Chitosan-film associated with mesenchymal stem cells enhanced regeneration of peripheral nerves: A rat sciatic nerve model

ObjectivePeripheral nerve injuries comprise significant portion of the nervous system injuries. Although peripheral nerves show some capacity of regeneration after injury, but the extent of regeneration is not remarkable. Regeneration might be through the activity of the mesenchymal stem cells (MSCs) which can release growth factors or extracellular matrix components or by the therapeutic effect of some material with the MSCs. The present study aimed to evaluate the regeneration of transected sciatic nerve by a therapeutic value of mesenchymal stem cells (MSCs) associated with chitosan-film (Cs) in rat. Materials & methodsMale Wistar rats (n=42, 180-200g) were randomly divided into intact; control; sham; Cs; MSCs; MSCs + Cs groups. Functional recovery was evaluated at 2, 4, 6 and 8 weeks after surgery using sciatic functional index (SFI), hot water paw immersion test, electrophysiological, histological analyses. ResultsThe rats in the MSCs+Cs group showed significant decrease in SFI and hot water paw immersion test during the 2nd to 8th weeks after surgery. Electrophysiological findings showed a significant decrease in latency time in the MSCs +Cs group. Amplitude of the nerve impulses also increased. Number of nerve fibers with more than 6 μm diameters increased significantly in MSCs+Cs. The number of nerve fibers with less than 4 μm diameters also increased significantly in MSCs+Cs group. ConclusionTaken together, mesenchymal stem cells associated with Cs could improve functional and histomorphological properties of the sciatic nerve after injury which may have some clinical outcomes as well.

Ultrastructural and histomorphologic properties of the internal thoracic artery: implications for coronary revascularization.

Coronary artery disease represents a major health problem worldwide for which coronary artery bypass surgery remains a standard of care. Among the several grafts that are available, the internal thoracic artery (ITA) has long been considered the best as several advantages have been described compared with other vessels (e.g. saphenous vein or radial artery), namely, an absent to minor atherosclerotic development. In fact, several studies showed the presence of preatherosclerotic lesions, such as intimal and/or medial thickening, medial fibrosis, among others, in the presence of certain cardiovascular risk factors as well as established atherosclerotic lesions (i.e. type II or more lesions). This paper primarily aimed at reviewing the current knowledge on the histomorphological characteristics of ITA as well as the comparative histomorphology of ITA with other vessel grafts currently in use in coronary surgery. As some of the evidence is not clear or consensual, this paper also aimed at reviewing the main histopathological, histomorphometrical, and ultrastructural findings in ITAs from patients with known cardiovascular risk factors (e.g. aging, obesity, hypertension, diabetes, smoking, and others). As the presence of preatherosclerotic and/or atherosclerotic lesions may compromise the success of the myocardial revascularization and lead to graft failure, contributing toward the associated morbidity and/or mortality, it is essential to improve the scientific knowledge on the structural characterization of ITAs and its correlation with the cardiovascular risk profile.

A biological tube technique for the repair of peripheral nerve defects using 'stuffed nerves'.

Presently described is research examining the "stuffed nerve" technique to repair peripheral nerve defects. Twenty-one male Wistar Albino rats were divided into 3 groups of 7, and standard 10-mm defects were created in the sciatic nerve of all subjects. Rats were treated with autogenous nerve graft (Group 1), hollow vein graft (Group 2), or vein graft stuffed with shredded nerves (Group 3). After 12 weeks, electrophysiological and histomorphological analyses were performed to evaluate axonal regeneration. Rat groups were compared in terms of latency period and peak-to-peak potential. Latency period was significantly shorter and peak-to-peak potential was significantly greater in Group 1 than in Group 2. However, latency period and peak-to-peak potential did not differ significantly between Groups 1 and 3 or between Groups 2 and 3. To evaluate axonal regeneration, number of axons, axon diameter and myelin sheath thickness was compared between groups. Results indicated that axonal regeneration was similar in Groups 1 and 3, and was better than results seen in Group 2. The stuffed nerve technique is an alternative to autogenous nerve grafting and produces similar electrophysiological and histomorphological properties.

A biomedical cell product for the regeneration of articular cartilage: Biocompatible and histomorphological properties (an experimental model of subcutaneous implantation)

An in vivo study of the biological safety of a biomedical cell product for regeneration of articular cartilage with subcutaneous implantation of cell-engineered cartilage tissue was carried out. A comparative study of the biological safety of the cell-engineered construct of human articular cartilage tissue (CEC hAT) and the biocompatible BMCH matrix in an implantation test did not show significant differences between the control and experimental groups. The results demonstrated that the cell component of the cell-engineered construct of hAT is not a risk factor for its implantation for up to 28 days. During the study, some signs of formation of articular tissue were observed at the site of subcutaneous implantation of the CEC hAT. Considering high proliferation and differentiation activity of the CEC hAT previously confirmed after its cultivation for 42 days, it is possible to suggest the potential of using CEC hAT for regeneration of injured cartilage as an injection form of biomedical cell product.

Neurons from the adult human dentate nucleus: Neural networks in the neuron classification

Topological (central vs. border neuron type) and morphological classification of adult human dentate nucleus neurons according to their quantified histomorphological properties using neural networks on real and virtual neuron samples. In the real sample 53.1% and 14.1% of central and border neurons, respectively, are classified correctly with total of 32.8% of misclassified neurons. The most important result present 62.2% of misclassified neurons in border neurons group which is even greater than number of correctly classified neurons (37.8%) in that group, showing obvious failure of network to classify neurons correctly based on computational parameters used in our study. On the virtual sample 97.3% of misclassified neurons in border neurons group which is much greater than number of correctly classified neurons (2.7%) in that group, again confirms obvious failure of network to classify neurons correctly. Statistical analysis shows that there is no statistically significant difference in between central and border neurons for each measured parameter (p>0.05). Total of 96.74% neurons are morphologically classified correctly by neural networks and each one belongs to one of the four histomorphological types: (a) neurons with small soma and short dendrites, (b) neurons with small soma and long dendrites, (c) neuron with large soma and short dendrites, (d) neurons with large soma and long dendrites. Statistical analysis supports these results (p<0.05). Human dentate nucleus neurons can be classified in four neuron types according to their quantitative histomorphological properties. These neuron types consist of two neuron sets, small and large ones with respect to their perykarions with subtypes differing in dendrite length i.e. neurons with short vs. long dendrites. Besides confirmation of neuron classification on small and large ones, already shown in literature, we found two new subtypes i.e. neurons with small soma and long dendrites and with large soma and short dendrites. These neurons are most probably equally distributed throughout the dentate nucleus as no significant difference in their topological distribution is observed.

A101 紫外線を照射した皮膚組織の組織形態学的特性(A1-1 生体計測1)

A101 紫外線を照射した皮膚組織の組織形態学的特性(A1-1 生体計測1)