Quantitative Image Analysis of Fibrillar Collagens Reveals Novel Diagnostic and Prognostic Biomarkers and Histotype-Dependent Aberrant Mechanobiology in Lung Cancer.

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pathology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE settings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve = 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC independently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (α-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff microenvironment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities.

In situ type I oligomeric collagen macroencapsulation promotes islet longevity and function in vitro and in vivo.

Widespread use of pancreatic islet transplantation for treatment of type 1 diabetes (T1D) is currently limited by requirements for long-term immunosuppression, limited donor supply, and poor long-term engraftment and function. Upon isolation from their native microenvironment, islets undergo rapid apoptosis, which is further exacerbated by poor oxygen and nutrient supply following infusion into the portal vein. Identifying alternative strategies to restore critical microenvironmental cues, while maximizing islet health and function, is needed to advance this cellular therapy. We hypothesized that biophysical properties provided through type I oligomeric collagen macroencapsulation are important considerations when designing strategies to improve islet survival, phenotype, and function. Mouse islets were encapsulated at various Oligomer concentrations (0.5 -3.0 mg/ml) or suspended in media and cultured for 14 days, after which viability, protein expression, and function were assessed. Oligomer-encapsulated islets showed a density-dependent improvement in in vitro viability, cytoarchitecture, and insulin secretion, with 3 mg/ml yielding values comparable to freshly isolated islets. For transplantation into streptozotocin-induced diabetic mice, 500 islets were mixed in Oligomer and injected subcutaneously, where rapid in situ macroencapsulation occurred, or injected with saline. Mice treated with Oligomer-encapsulated islets exhibited rapid (within 24 h) diabetes reversal and maintenance of normoglycemia for 14 (immunocompromised), 90 (syngeneic), and 40 days (allogeneic). Histological analysis showed Oligomer-islet engraftment with maintenance of islet cytoarchitecture, revascularization, and no foreign body response. Oligomer-islet macroencapsulation may provide a useful strategy for prolonging the health and function of cultured islets and has potential as a subcutaneous injectable islet transplantation strategy for treatment of T1D.

Fibrillar collagen injection for organ protection during thermal ablation of hepatic malignancies.

Percutaneous image-guided ablation is performed throughout many areas of the body for various pathologies including hepatic malignancies. Heat and cold-based ablative technologies are effective and well-tolerated with an acceptable safety profile. However, ablative therapies may be technically more challenging and cause collateral thermal injury if the targeted lesion is adjacent to critical organs. Previously, techniques including artificial ascites and pneumoperitoneum have been utilized to displace or insulate critical structures from the ablation zone. This technical innovation describes (10-30 mL) fibrillar collagen dissolved in fluid as a focal thermal insulation technique. Small volume fibrillar collagen instillation, and thermal ablation were technically successful in three cases without complication. Clinical follow-up and 3-month imaging confirmed complete ablation of all hepatic malignancies without collateral injury.

Matrix-Induced Autologous Chondrocyte Implantation (MACI) grafting for osteochondral lesions of the talus.

BACKGROUND: Articular cartilage is limited in its ability to repair itself. Matrix-induced Autologous Chondrocyte Implantation (MACI) is an established treatment method for such articular cartilage defects in the knee. Recently the technique has been used in the ankle. We present a series of patients treated with MACI for osteochondral defects of the ankle, and assess the functional and clinical results. MATERIALS AND METHODS: From August 2003 to February 2006, 20 patients underwent MACI grafting for osteochondral defects in the ankle. Age ranged from 19 to 61 (mean, 36) years. Mean followup was 21.1 months. Clinical and functional evaluations were conducted using the AOFAS scoring system. RESULTS: The mean size was 233 mm(2). There was a significant improvement in mean AOFAS score from 60 (range, 25 to 87) to 87 (range, 41 to 100) (p < 0.0001). Overall improvement in pain scores was also significant (p < 0.0001). All osteotomies healed. Four patients required hardware removal and two underwent arthroscopic debridement for anterior impingement. There were two failures which are awaiting subsequent procedures. CONCLUSION: We believe MACI is a reliable treatment method for talar osteochondral defects. The method usually requires an intra-articular osteotomy, although this proved to be a reasonably simple aspect of the procedure for the treatment of cartilage defects of the talus.

Smoking and nicotine exposure delay development of collagen-induced arthritis in mice.

INTRODUCTION: Recent epidemiologic studies have implicated smoking as an environmental risk factor for the development of rheumatoid arthritis (RA). The aim of the present study is the evaluation of the role of cigarette smoke (CS) in the pathogenesis of collagen-induced arthritis in mice. METHODS: DBA/1 mice exposed to CS for 16 weeks (n = 25) and mice exposed to nicotine in drinking water (n = 10) were immunized with collagen type II (CII). Severity of arthritis was evaluated clinically and morphologically and compared with control mice (n = 35). Intensity of inflammation was evaluated by serum IL-6 and TNF-alpha levels. Additionally, antibody response to CII (anti-CII) and citrullinated peptides (aCCP) was measured. RESULTS: Clinical evaluation of arthritis showed a delayed onset of arthritis in CS-exposed mice compared with non-smoking controls (P < 0.05). Histologic index and weight changes were comparable between the groups; however, smoking mice presented less weight loss during the acute phase of the disease and gained weight significantly faster in the recovery phase (P < 0.05). Similar results were obtained in the mice exposed to nicotine. Nicotine also showed a direct anti-inflammatory effect diminishing IL-6 production by stimulated splenocytes in vitro (P < 0.001). Additionally, smoking mice had lower levels of aCCP and anti-CII antibodies compared with non-smoking (P < 0.05). CONCLUSIONS: Neither smoking nor nicotine exposure aggravates development of CII-induced arthritis in mouse model. Moreover, CS exposure was associated with a lower level of anti-CII antibodies, providing a possible explanation for a delay of arthritis onset in this group.

A elasticidade do ligamento colateral medial da articulação do cotovelo de cão não advém de elastina / The elasticity of the medial collateral ligament of the canine elbow joint does not come from elastin

A literatura relata que ligamentos consistem de tecido conjuntivo denso, composto por água, colágeno tipos I e III, diversas proteoglicanas, pouca elastina e várias outras substâncias. Além disso, os ligamentos, quando testados in vitro com tensão longitudinal e unidirecional, apresentam um comportamento mecânico não-linear, ou seja, as fibras colágenas são alongadas aos poucos, perdendo seu padrão ondulado, até que todas estejam no limite máximo de tração e iniciem o rompimento. Portanto, no presente estudo avaliou-se a presença de fibras elásticas (elastina) no ligamento colateral medial do cotovelo de cães adultos para ponderar se a elasticidade do referido ligamento deve-se à presença de fibras elásticas ou às propriedades elásticas do colágeno ou à combinação de ambas. Foram utilizadas quatro articulações, de machos e fêmeas em igual proporção, das quais foram adquiridas as amostras das porções médias dos ligamentos colaterais mediais para a rotina histológica. Os cortes foram corados pela técnica de Weigert, e não foi observada a presença de fibras elásticas, detectável por esta técnica à microscopia de luz. Concluiu-se que a elasticidade do ligamento colateral medial do cotovelo de cão deve-se, principalmente, ao padrão ondulado das fibras colágenas, devido à quantidade ínfima ou até à inexistência de fibras elásticas nesta estrutura.

The literature reports that ligaments consist of connective tissue, composed by water, Type I and III collagen, several proteoglycans, some elastin and other substances. Ligaments tested in vitro with longitudinal and unidirectional tension exhibit non-linear mechanical behavior; the collagen fibers are stretched little by little, losing their undulating pattern, until reaching the maximum limit of traction and failure begins. The aim of the present study was to assess the presence of elastin in the medial collateral ligament of the elbow in adult dogs to determine whether the stretching of this ligament is due to the presence of elastic fibers, the elastic property of the collagen or the combination of both. Four joints were used from males and females in equal proportion, taking the medial collateral ligaments for the histological examination. To detect the presence of elastic fibers, sections were stained using the Weigert method. However, light microscopy revealed no elastic fibers. It was concluded that the elasticity of the canine elbow medial collateral ligament is mainly due to the undulated pattern of the collagen fibers, considering the trace amount or inexistence of elastic fibers in this structure.

Evaluation of autologous platelet-poor plasma gel as a hemostatic adjunct after posterior iliac crest bone harvest.

PURPOSE: To evaluate the hemostatic efficacy of autologous platelet-poor plasma (PPP) gel following posterior iliac crest bone graft harvesting for oral and maxillofacial reconstruction. PATIENTS AND METHODS: This was a prospective study of 24 consecutive patients involving 26 posterior iliac crest bone harvests that had bone wax and either 1-gram of bovine microfibrillar collagen or 20 mL of autologous PPP, activated as a gel, used as adjunct hemostatic agents. Compression bulb suction drain was placed into the graft site and drain output recorded every 8 hours for 64 hours. Cost analysis was also undertaken between the 2 methods. Statistical significance between means of each group was determined by Student's t test and found significant for P < .05. RESULTS: There were no statistically significant differences in average drain output between the PPP and MFC groups for each 8-hour interval. There was no statically significant difference in average total drain output between the PPP and MFC groups over the entire 64 hour period. Additionally, unlike the addition of MFC, the addition of PPP added no additional costs to the procedure. CONCLUSION: PPP gel, when compared with bovine microfibrillar collagen, is an effective and inexpensive adjunct in hemostasis following posterior iliac crest bone harvest.

Orthopaedic tissue engineering: from laboratory to the clinic.

Tissue engineering involves the use of cells (either adult, mesenchymal or embryonic stem cells) coupled with biological or artificial matrices or scaffolds which guide the cells during repair or regeneration of the tissue. Recently discovered and isolated growth factors can promote either adult or stem-cell growth and differentiation along selected pathways to re-form and repair skeletal tissues in adults. Bone repair enhancement and replacement is now possible with the use of tissue-engineering technologies. It is now possible to repair articular cartilage using the patient's own articular chondrocytes retrieved during arthroscopy, and expanded in vitro. Clinical results of this technique are very satisfactory.