Liver click dECM hydrogels for engineering hepatic microenvironments.

Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael-type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of l-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young's moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel's mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver's natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications.

In vivo study to assess fat embolism resulting from the Reamer-Irrigator-Aspirator 2 system compared to a novel aspirator-based concept for intramedullary bone graft harvesting.

INTRODUCTION: Fat embolism (FE) following intramedullary (IM) reaming can cause severe pulmonary complications and sudden death. Recently, a new harvesting concept was introduced in which a novel aspirator is used first for bone marrow (BM) aspiration and then for subsequent aspiration of morselized endosteal bone during sequential reaming (A + R + A). In contrast to the established Reamer-Irrigator-Aspirator (RIA) 2 system, the new A + R + A concept allows for the evacuation of fatty BM prior to reaming. In this study, we hypothesized that the risk of FE, associated coagulopathic reactions and pulmonary FE would be comparable between the RIA 2 system and the A + R + A concept. MATERIALS AND METHODS: Intramedullary bone graft was harvested from intact femora of 16 Merino sheep (age: 1-2 years) with either the RIA 2 system (n = 8) or the A + R + A concept (n = 8). Fat intravasation was monitored with the Gurd test, coagulopathic response with D-dimer blood level concentration and pulmonary FE with histological evaluation of the lungs. RESULTS: The total number and average size of intravasated fat particles was similar between groups (p = 0.13 and p = 0.98, respectively). D-dimer concentration did not significantly increase within 4 h after completion of surgery (RIA 2: p = 0.82; A + R + A: p = 0.23), with an interaction effect similar between groups (p = 0.65). The average lung area covered with fat globules was similar between groups (p = 0.17). CONCLUSIONS: The use of the RIA 2 system and the novel A + R + A harvesting concept which consists of BM evacuation followed by sequential IM reaming and aspiration of endosteal bone, resulted in only minor fat intravasation, coagulopathic reactions and pulmonary FE, with no significant differences between the groups. Our results, therefore, suggest that both the RIA 2 system and the new A + R + A concept are comparable technologies in terms of FE-related complications.

GelMA and Biomimetic Culture Allow the Engineering of Mineralized, Adipose, and Tumor Tissue Human Microenvironments for the Study of Advanced Prostate Cancer In Vitro and In Vivo.

Increasing evidence shows bone marrow (BM)-adipocytes as a potentially important contributor in prostate cancer (PCa) bone metastases. However, a lack of relevant models has prevented the full understanding of the effects of human BM-adipocytes in this microenvironment. It is hypothesized that the combination of tunable gelatin methacrylamide (GelMA)-based hydrogels with the biomimetic culture of human cells would offer a versatile 3D platform to engineer human bone tumor microenvironments containing BM-adipocytes. Human osteoprogenitors, adipocytes, and PCa cells are individually cultured in vitro in GelMA hydrogels, leading to mineralized, adipose, and PCa tumor 3D microtissues, respectively. Osteoblast mineralization and tumor spheroid formation are tailored by hydrogel stiffness with lower stiffnesses correlating with increased mineralization and tumor spheroid size. Upon coculture with tumor cells, BM-adipocytes undergo morphological changes and delipidation, suggesting reciprocal interactions between the cell types. When brought in vivo, the mineralized and adipose microtissues successfully form a humanized fatty bone microenvironment, presenting, for the first time, with human adipocytes. Using this model, an increase in tumor burden is observed when human adipocytes are present, suggesting that adipocytes support early bone tumor growth. The advanced platform presented here combines natural aspects of the microenvironment with tunable properties useful for bone tumor research.

The Challenges and Emerging Opportunities of Targeting Cytokines and Chemokine-Driven Inflammatory Signals in Metastatic Castrate-Resistant Prostate Cancer.

Inflammation is a key risk factor and functional driver in the initiation and progression of prostate cancer (PCa). De-regulated cytokine and chemokine signaling facilitates critical communication between tumor cells and multiple cell lineages within the tumor microenvironment (TME). Historical attempts at using targeted approaches to disrupt inflammation have been disappointing, with sub-optimal or negligible clinical benefit. Our increased awareness of the myeloid infiltrate in supporting the acquisition of castrate resistance and underpinning the abject response of advanced PCa to immunotherapy has re-focused attention on improved strategies to disrupt these complex cytokine and chemokine signaling networks within the TME. These ongoing and prospective strategies are principally focused on employing cytokine-/chemokine-directed therapies in informed combination with androgen signaling inhibitors or immunotherapeutic agents and, increasingly, with due consideration of the genetic context of the tumor. The availability of molecular-targeted therapeutic agents directed against the critical signal transduction nodes activated by cytokine and chemokine signaling in tumor cells provides opportunities to reduce the impacts of biological redundancy. Precision-based trials that deploy this latest generation of cytokine- and chemokine-directed therapeutics, directed to enriched patient cohorts in a biologically informed and biomarker-guided manner, have the potential to diversify the armamentarium of agents that is required in order to transform long-term outcomes for a currently incurable and genetically heterogenous disease.

A humanised rat model of osteosarcoma reveals ultrastructural differences between bone and mineralised tumour tissue.

Current xenograft animal models fail to accurately replicate the complexity of human bone disease. To gain translatable and clinically valuable data from animal models, new in vivo models need to be developed that mimic pivotal aspects of human bone physiology as well as its diseased state. Above all, an advanced bone disease model should promote the development of new treatment strategies and facilitate the conduction of common clinical interventional procedures. Here we describe the development and characterisation of an orthotopic humanised tissue-engineered osteosarcoma (OS) model in a recently genetically engineered x-linked severe combined immunodeficient (X-SCID) rat. For the first time in a genetically modified rat, our results show the successful implementation of an orthotopic humanised tissue-engineered bone niche supporting the growth of a human OS cell line including its metastatic spread to the lung. Moreover, we studied the inter- and intraspecies differences in ultrastructural composition of bone and calcified tissue produced by the tumour, pointing to the crucial role of humanised animal models.

A humanized orthotopic tumor microenvironment alters the bone metastatic tropism of prostate cancer cells.

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, and bone is the most frequent site of metastasis. The tumor microenvironment (TME) impacts tumor growth and metastasis, yet the role of the TME in PCa metastasis to bone is not fully understood. We used a tissue-engineered xenograft approach in NOD-scid IL2Rγnull (NSG) mice to incorporate two levels of humanization; the primary tumor and TME, and the secondary metastatic bone organ. Bioluminescent imaging, histology, and immunohistochemistry were used to study metastasis of human PC-3 and LNCaP PCa cells from the prostate to tissue-engineered bone. Here we show pre-seeding scaffolds with human osteoblasts increases the human cellular and extracellular matrix content of bone constructs, compared to unseeded scaffolds. The humanized prostate TME showed a trend to decrease metastasis of PC-3 PCa cells to the tissue-engineered bone, but did not affect the metastatic potential of PCa cells to the endogenous murine bones or organs. On the other hand, the humanized TME enhanced LNCaP tumor growth and metastasis to humanized and murine bone. Together this demonstrates the importance of the TME in PCa bone tropism, although further investigations are needed to delineate specific roles of the TME components in this context.

A Preclinical Animal Model for the Study of Scaffold-Guided Breast Tissue Engineering.

Scaffold-guided breast tissue engineering (SGBTE) has the potential to transform reconstructive breast surgery. Currently, there is a deficiency in clinically relevant animal models suitable for studying novel breast tissue engineering concepts. To date, only a small number of large animal studies have been conducted and characterization of these large animal models is poorly described in the literature. Addressing this gap in the literature, this publication comprehensively describes our original porcine model based on the current published literature and the experience gained from previous animal studies conducted by our research group. In a long-term experiment using our model, we investigated our SGBTE approach by implanting 60 additively manufactured bioresorbable scaffolds under the panniculus carnosus muscle along the flanks of 12 pigs over 12 months. Our model has the flexibility to compare multiple treatment modalities where we successfully investigated scaffolds filled with various treatments of immediate and delayed fat graft and augmentation with platelet rich plasma. No wound complications were observed using our animal model. We were able to grow clinically relevant volumes of soft tissue, which validates our model. Our preclinical large animal model is ideally suited to assess different scaffold or hydrogel-driven soft tissue regeneration strategies. Impact statement The ability to regenerate soft tissue through scaffold-guided tissue engineering concepts can transform breast reconstructive surgery. We describe an original preclinical large animal model to study controlled and reproducible scaffold-guided breast tissue engineering (SGBTE) concepts. This model features the flexibility to investigate multiple treatment conditions per animal, making it an efficient model. We have validated our model with a long-term experiment over 12 months, which exceeds other shorter published studies. Our SGBTE concept provides a more clinically relevant approach in terms of breast reconstruction. Future studies using this model will support the translation of SGBTE into clinical practice.

A 3D-printed biomaterials-based platform to advance established therapy avenues against primary bone cancers.

In this study we developed and validated a 3D-printed drug delivery system (3DPDDS) to 1) improve local treatment efficacy of commonly applied chemotherapeutic agents in bone cancers to ultimately decrease their systemic side effects and 2) explore its concomitant diagnostic potential. Thus, we locally applied 3D-printed medical-grade polycaprolactone (mPCL) scaffolds loaded with Doxorubicin (DOX) and measured its effect in a humanized primary bone cancer model. A bioengineered species-sensitive orthotopic humanized bone niche was established at the femur of NOD-SCID IL2Rγnull (NSG) mice. After 6 weeks of in vivo maturation into a humanized ossicle, Luc-SAOS-2 cells were injected orthotopically to induce local growth of osteosarcoma (OS). After 16 weeks of OS development, a biopsy-like defect was created within the tumor tissue to locally implant the 3DPDDS with 3 different DOX loading doses into the defect zone. Histo- and morphological analysis demonstrated a typical invasive OS growth pattern inside a functionally intact humanized ossicle as well as metastatic spread to the murine lung parenchyma. Analysis of the 3DPDDS revealed the implants' ability to inhibit tumor infiltration and showed local tumor cell death adjacent to the scaffolds without any systemic side effects. Together these results indicate a therapeutic and diagnostic capacity of 3DPDDS in an orthotopic humanized OS tumor model.

Engineering a Humanised Niche to Support Human Haematopoiesis in Mice: Novel Opportunities in Modelling Cancer.

Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour-microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.

Correction: Humanized bone facilitates prostate cancer metastasis and recapitulates therapeutic effects of Zoledronic acid in vivo.

[This corrects the article DOI: 10.1038/s41413-019-0072-9.].

Mobilizing serum factors and immune cells through exercise to counteract age-related changes in cancer risk.

An increasing body of evidence suggests that age-related immune changes and chronic inflammation contribute to cancer development. Recognizing that exercise has protective effects against cancer, promotes immune function, and beneficially modulates inflammation with ageing, this review outlines the current evidence indicating an emerging role for exercise immunology in preventing and treating cancer in older adults. A specific focus is on data suggesting that muscle- derived cytokines (myokines) mediate anti-cancer effects through promoting immunosurveillance against tumourigenesis or inhibiting cancer cell viability. Previous studies suggested that the exercise-induced release of myokines and other endocrine factors into the blood increases the capacity of blood serum to inhibit cancer cell growth in vitro. However, little is known about whether this effect is influenced by ageing. Prostate cancer is the second most common cancer in men. We therefore examined the effects of serum collected before and after exercise from healthy young and older men on the metabolic activity of androgen-responsive LNCaP and androgen-unresponsive PC3 prostate cancer cells. Exercise-conditioned serum collected from the young group did not alter cell metabolic activity, whereas post-exercise serum (compared with pre-exercise serum) from the older men inhibited the metabolic activity of LNCaP cancer cells. Serum levels of candidate cancer-inhibitory myokines oncostatin M and osteonectin increased in both age groups following exercise. Serum testosterone increased only in the younger men postexercise, potentially attenuating inhibitory effects of myokines on the LNCaP cell viability. The data from our study and the evidence in this review suggest that mobilizing serum factors and immune cells may be a key mechanism of how exercise counteracts cancer in the older population.

Targeted camptothecin delivery via silicon nanoparticles reduces breast cancer metastasis.

In advanced breast cancer (BCa) patients, not the primary tumor, but the development of distant metastases, which occur mainly in the organ bone, and their adverse health effects are responsible for high mortality. Targeted delivery of already known drugs which displayed potency, but rather unfavorable pharmacokinetic properties, might be a promising approach to overcome the current limitations of metastatic BCa therapy. Camptothecin (CPT) is a highly cytotoxic chemotherapeutic compound, yet poorly water-soluble and non-specific. Here, CPT was loaded into porous silicon nanoparticles (pSiNP) displaying the epidermal growth factor receptor (EGFR)-targeting antibody (Ab) cetuximab to generate a soluble and targeted nanoscale delivery vehicle for cancer treatment. After confirming the cytotoxic effect of targeted CPT-loaded pSiNP in vitro on MDA-MB-231BO cells, nanoparticles were studied in a humanized BCa bone metastasis mouse model. Humanized tissue-engineered bone constructs (hTEBCs) provided a humanized microenvironment for BCa bone metastases in female NOD-scid IL2Rgnull (NSG) mice. Actively targeted CPT-loaded pSiNP led to a reduction of orthotopic primary tumor growth, increased survival rate and significant decrease in hTEBC and murine lung, liver and bone metastases. This study demonstrates that targeted delivery via pSiNP is an effective approach to employ CPT and other potent anti-cancer compounds with poor pharmacokinetic profiles in cancer therapy.

A preclinical large-animal model for the assessment of critical-size load-bearing bone defect reconstruction.

Critical-size bone defects, which require large-volume tissue reconstruction, remain a clinical challenge. Bone engineering has the potential to provide new treatment concepts, yet clinical translation requires anatomically and physiologically relevant preclinical models. The ovine critical-size long-bone defect model has been validated in numerous studies as a preclinical tool for evaluating both conventional and novel bone-engineering concepts. With sufficient training and experience in large-animal studies, it is a technically feasible procedure with a high level of reproducibility when appropriate preoperative and postoperative management protocols are followed. The model can be established by following a procedure that includes the following stages: (i) preoperative planning and preparation, (ii) the surgical approach, (iii) postoperative management, and (iv) postmortem analysis. Using this model, full results for peer-reviewed publication can be attained within 2 years. In this protocol, we comprehensively describe how to establish proficiency using the preclinical model for the evaluation of a range of bone defect reconstruction options.

Humanized bone facilitates prostate cancer metastasis and recapitulates therapeutic effects of zoledronic acid in vivo.

Advanced prostate cancer (PCa) is known for its high prevalence to metastasize to bone, at which point it is considered incurable. Despite significant effort, there is no animal model capable of recapitulating the complexity of PCa bone metastasis. The humanized mouse model for PCa bone metastasis used in this study aims to provide a platform for the assessment of new drugs by recapitulating the human-human cell interactions relevant for disease development and progression. The humanized tissue-engineered bone construct (hTEBC) was created within NOD-scid IL2rgnull (NSG) mice and was used for the study of experimental PC3-Luc bone metastases. It was confirmed that PC3-Luc cells preferentially grew in the hTEBC compared with murine bone. The translational potential of the humanized mouse model for PCa bone metastasis was evaluated with two clinically approved osteoprotective therapies, the non-species-specific bisphosphonate zoledronic acid (ZA) or the human-specific antibody Denosumab, both targeting Receptor Activator of Nuclear Factor Kappa-Β Ligand. ZA, but not Denosumab, significantly decreased metastases in hTEBCs, but not murine femora. These results highlight the importance of humanized models for the preclinical research on PCa bone metastasis and indicate the potential of the bioengineered mouse model to closely mimic the metastatic cascade of PCa cells to human bone. Eventually, it will enable the development of new effective antimetastatic treatments.

A humanized bone microenvironment uncovers HIF2 alpha as a latent marker for osteosarcoma.

The quest for predictive tumor markers for osteosarcoma (OS) has not well progressed over the last two decades due to a lack of preclinical models. The aim of this study was to investigate if microenvironmental modifications in an original humanized in vivo model alter the expression of OS tumor markers. Human bone micro-chips and bone marrow, harvested during hip arthroplasty, were implanted at the flanks of NOD/scid mice. We administered recombinant human bone morphogenetic protein 7 (rhBMP-7) in human bone micro-chips/bone marrow group I in order to modulate bone matrix and bone marrow humanization. Ten weeks post-implantation, human Luc-SAOS-2 OS cells were injected into the humanized tissue-engineered bone organs (hTEBOs). Tumors were harvested 5 weeks post-implantation to determine the expression of the previously described OS markers ezrin, periostin, VEGF, HIF1α and HIF2α. Representation of these proteins was analyzed in two different OS patient cohorts. Ezrin was downregulated in OS in hTEBOs with rhBMP-7, whereas HIF2α was significantly upregulated in comparison to hTEBOs without rhBMP-7. The expression of periostin, VEGF and HIF1α did not differ significantly between both groups. HIF2α was consistently present in OS patients and dependent on tumor site and clinical stage. OS patients post-chemotherapy had suppressed levels of HIF2α. In conclusion, we demonstrated the overall expression of OS-related factors in a preclinical model, which is based on a humanized bone organ. Our preclinical research results and analysis of two comprehensive patient cohorts imply that HIF2α is a potential prognostic marker and/or therapeutic target. STATEMENT OF SIGNIFICANCE: This study demonstrates the clinical relevance of the humanized organ bone microenvironment in osteosarcoma research and validates the expression of tumor markers, especially HIF2α. The convergence of clinically proven bone engineering concepts for the development of humanized mice models is a new starting point for investigations of OS-related marker expression. The validation and first data set in such a model let one conclude that further clinical studies on the role of HIF2α as a prognostic marker and its potential as therapeutic target is a condition sine qua non.

Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair.

Repair after damage is essential for tissue homeostasis. Postmenstrual endometrial repair is a cyclical manifestation of rapid, scar-free, tissue repair taking ∼3-5 d. Skin repair after wounding is slower (∼2 wk). In the case of chronic wounds, it takes months to years to restore integrity. Herein, the unique "rapid-repair" endometrial environment is translated to the "slower repair" skin environment. Menstrual fluid (MF), the milieu of postmenstrual endometrial repair, facilitates healing of endometrial and keratinocyte "wounds" in vitro, promoting cellular adhesion and migration, stimulates keratinocyte migration in an ex vivo human skin reconstruct model, and promotes re-epithelialization in an in vivo porcine wound model. Proteomic analysis of MF identified a large number of proteins: migration inhibitory factor, neutrophil gelatinase-associated lipocalin, follistatin like-1, chemokine ligand-20, and secretory leukocyte protease inhibitor were selected for further investigation. Functionally, they promote repair of endometrial and keratinocyte wounds by promoting migration. Translation of these and other MF factors into a migration-inducing treatment paradigm could provide novel treatments for tissue repair.-Evans, J., Infusini, G., McGovern, J., Cuttle, L., Webb, A., Nebl, T., Milla, L., Kimble, R., Kempf, M., Andrews, C. J., Leavesley, D., Salamonsen, L. A. Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair.

Humanization of the Prostate Microenvironment Reduces Homing of PC3 Prostate Cancer Cells to Human Tissue-Engineered Bone.

The primary tumor microenvironment is inherently important in prostate cancer (PCa) initiation, growth and metastasis. However, most current PCa animal models are based on the injection of cancer cells into the blood circulation and bypass the first steps of the metastatic cascade, hence failing to investigate the influence of the primary tumor microenvironment on PCa metastasis. Here, we investigated the spontaneous metastasis of PC3 human PCa cells from humanized prostate tissue, containing cancer-associated fibroblasts (CAFs) and prostate lymphatic and blood vessel endothelial cells (BVEC), to humanized tissue-engineered bone constructs (hTEBC) in NOD-SCID IL2Rγnull (NSG) mice. The hTEBC formed a physiologically mature organ bone which allowed homing of metastatic PCa cells. Humanization of prostate tissue had no significant effect on the tumor burden at the primary site over the 4 weeks following intraprostatic injection, yet reduced the incidence and burden of metastases in the hTEBC. Spontaneous PCa metastases were detected in the lungs and spleen with no significant differences between the humanized and non-humanized prostate groups. A significantly greater metastatic tumor burden was observed in the liver when metastasis occurred from the humanized prostate. Together, our data suggests that the presence of human-derived CAFs and BVECs in the primary PCa microenvironment influences selectively the metastatic and homing behavior of PC3 cells in this model. Our orthotopic and humanized prostate cancer model developed via convergence of cancer research and tissue engineering concepts provides an important platform to study species-specific PCa bone metastasis and to develop and test therapeutic strategies.

Non-linear optical microscopy and histological analysis of collagen, elastin and lysyl oxidase expression in breast capsular contracture.

BACKGROUND: Capsular contracture is one of the most common complications in surgical interventions for aesthetic breast augmentation or post-mastectomy breast reconstruction involving the use of silicone prostheses. Although the precise cause of capsular contracture is yet unknown, the leading hypothesis is that it is caused by long-term unresolved foreign body reaction towards the silicone breast implant. To authors' best knowledge, this is the first study that elucidates the presence of lysyl oxidase (LOX)-an enzyme that is involved in collagen and elastin crosslinking within fibrous capsules harvested from patients with severe capsular contracture. It was hypothesized that over-expression of LOX plays a role in the irreversible crosslinking of collagen and elastin which, in turn, stabilizes the fibrous proteins and contributes to the progression of capsular contracture. METHODS: Eight fibrous capsules were collected from patients undergoing capsulectomy procedure, biomechanical testing was performed for compressive Young's moduli and evaluated for Type I and II collagen, elastin and LOX by means of non-linear optical microscopy and immunohistology techniques. RESULTS: Observations revealed the heterogeneity of tissue structure within and among the collected fibrous capsules. Regardless of the tissue structure, it has been shown that LOX expression was intensified at the implant-to-tissue interface. CONCLUSION: Our results indicate the involvement of LOX in the initiation of fibrous capsule formation which ultimately contributes towards the progression of capsular contracture.

Immune system augmentation via humanization using stem/progenitor cells and bioengineering in a breast cancer model study.

Despite significant advances, most current in vivo models fail to fully recapitulate the biological processes that occur in humans. Here we aimed to develop an advanced humanized model with features of an organ bone by providing different bone tissue cellular compartments including preosteoblasts, mesenchymal stem/stromal (MSCs), endothelial and hematopoietic cells in an engineered microenvironment. The bone compartment was generated by culturing the human MSCs, umbilical vein endothelial cells with gelatin methacryloyl hydrogels in the center of a melt-electrospun polycaprolactone tubular scaffolds, which were seeded with human preosteoblasts. The tissue engineered bone (TEB) was subcutaneously implanted into the NSG mice and formed a morphologically and functionally organ bone. Mice were further humanized through the tail vein injection of human cord blood derived CD34+ cells, which then populated in the mouse bone marrow, spleen and humanized TEB (hTEB). 11 weeks after CD34+ transplantation, metastatic breast cancer cells (MDA-MB-231BO) were orthotopically injected. Cancer cell injection resulted in the formation of a primary tumor and metastasis to the hTEB and mouse organs. Less frequent metastasis and lower tumor burden were observed in hematochimeric mice, suggesting an immune-mediated response against the breast cancer cells. Overall, our results demonstrate the efficacy of tissue engineering approaches to study species-specific cancer-bone interactions. Further studies using genetically modified hematopoietic stem cells and bioengineered microenvironments will enable us to address the specific roles of signaling molecules regulating hematopoietic niches and cancer metastasis in vivo.

Animal models for bone tissue engineering and modelling disease.

Tissue engineering and its clinical application, regenerative medicine, are instructing multiple approaches to aid in replacing bone loss after defects caused by trauma or cancer. In such cases, bone formation can be guided by engineered biodegradable and nonbiodegradable scaffolds with clearly defined architectural and mechanical properties informed by evidence-based research. With the ever-increasing expansion of bone tissue engineering and the pioneering research conducted to date, preclinical models are becoming a necessity to allow the engineered products to be translated to the clinic. In addition to creating smart bone scaffolds to mitigate bone loss, the field of tissue engineering and regenerative medicine is exploring methods to treat primary and secondary bone malignancies by creating models that mimic the clinical disease manifestation. This Review gives an overview of the preclinical testing in animal models used to evaluate bone regeneration concepts. Immunosuppressed rodent models have shown to be successful in mimicking bone malignancy via the implantation of human-derived cancer cells, whereas large animal models, including pigs, sheep and goats, are being used to provide an insight into bone formation and the effectiveness of scaffolds in induced tibial or femoral defects, providing clinically relevant similarity to human cases. Despite the recent progress, the successful translation of bone regeneration concepts from the bench to the bedside is rooted in the efforts of different research groups to standardise and validate the preclinical models for bone tissue engineering approaches.