Tissue‐engineered nanoclay‐based 3D in vitro breast cancer model for studying breast cancer metastasis to bone

Breast cancer (BrCa) preferentially spreads to bone and colonises within the bone marrow to cause bone metastases. To improve the outcome of patients with BrCa bone metastasis, we need to understand better the mechanisms underlying bone metastasis. Researchers have relied heavily upon in vivo xenografts due to limited availability of human bone metastasis samples. A significant limitation of these is that they do not have a human bone microenvironment. To address this issue, we have developed a nanoclay‐based 3D in vitro model of BrCa bone metastasis using human mesenchymal stem cells (MSCs) and human BrCa cells mimicking late stage of BrCa pathogenesis at the metastatic site. This 3D model can provide a microenvironment suitable for cell–cell and cell–matrix interactions whilst retaining the behaviour of BrCa cells with different metastasis potential (i.e., highly metastatic MDA‐MB‐231 and low metastatic MCF‐7) as shown by the production of alkaline phosphatase and matrix metalloproteinase‐9. The sequential culture of MSCs with MCF‐7 exhibited 3D tumouroids formation and also occurrence of mesenchymal to epithelial transition of cancer metastasis as evidenced by gene expression and immunocytochemistry. The unique and distinct behaviour of highly metastatic MDA‐MB‐231 and the low metastatic MCF‐7 was observed at the bone metastasis site. The changes to migratory capabilities and invasiveness in MDA‐MB‐231 in comparison with tumour growth with MCF‐7 was observed. Together, a novel bone‐mimetic 3D in vitro BrCa model has been developed that could be used to study mechanisms governing the later stage of cancer pathogenesis in bone.     

The effect of wicking fibres in tissue‐engineered bone scaffolds

The major limitation of large tissue‐engineered constructs used for bone regeneration is the lack of vasculature and, therefore, lack of transport of essential nutrients, chemical factors and progenitor cells. Research approaches to improve the transport properties of large scaffolds focus on using angiogenic factors and vasculogenic cells to create new vasculature; however, the slow rate of vessel formation and reliance on vessel self‐assembly in these approaches is problematic. In this study, a novel approach has been proposed, using proprietary engineered ‘wicking’ fibres of non‐circular cross‐section that provide highly efficient transport for fluid and cells. The effect of wicking fibres on the movement of fluorescein isothiocyanate (FITC)‐conjugated protein in a three‐dimensional (3D) hydrogel system was analysed. The results indicated that the rate of diffusion of the fluorescent protein was greatly enhanced in hydrogels that contained wicking fibres in comparison to those that did not. The movement of progenitor cells along wicking fibres and round fibres was assessed. This study demonstrated that wicking fibres enhance the movement of critical growth factors and progenitor cells central for bone regeneration. The results suggested that the incorporation of wicking fibres into large tissue‐engineered constructs may improve the transport of growth factors and progenitor cells essential for bone formation. Copyright © 2014 John Wiley & Sons, Ltd.     

Axially vascularized tissue‐engineered bone constructs retain their in vivo angiogenic and osteogenic capacity after high‐dose irradiation

In order to introduce bone tissue engineering to the field of oncological reconstruction, we are investigating for the first time the effect of various doses of ionizing irradiation on axially vascularized bone constructs. Synthetic bone constructs were created and implanted in 32 Lewis rats. Each construct was axially vascularized through an arteriovenous loop made by direct anastomosis of the saphenous vessels. After 2 weeks, the animals received ionizing irradiation of 9 Gy, 12 Gy and 15 Gy, and were accordingly classified to groups I, II and III, respectively. Group IV was not irradiated and acted as a control. Tissue generation, vascularity, cellular proliferation and apoptosis were investigated either 2 or 5 weeks after irradiation through micro‐computed tomography, histomorphometry and real‐time polymerase chain reaction (PCR). At 2 weeks after irradiation, tissue generation and central vascularity were significantly lower and apoptosis was significantly higher in groups II and III than group IV, but without signs of necrosis. Cellular proliferation was significantly lower in groups I and II. After 5 weeks, the irradiated groups showed improvement in all parameters in relation to the control group, indicating a retained capacity for angiogenesis after irradiation. PCR results confirmed the expression of osteogenesis‐related genes in all irradiated groups. Dense collagen was detected 5 weeks after irradiation, and one construct showed discrete islands of bone indicating a retained osteogenic capacity after irradiation. This demonstrates for the first time that axial vascularization was capable of supporting a synthetic bone construct after a high dose of irradiation that is comparable to adjuvant radiotherapy. Copyright © 2016 John Wiley & Sons, Ltd.     

Three‐dimensional assembly of tissue‐engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)‐terephthalate–poly(butylene terephthalate) (PEGT–PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold‐seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet‐assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone. Copyright © 2013 John Wiley & Sons, Ltd.     

Scaffold–cell bone engineering in a validated preclinical animal model: precursors vs differentiated cell source

The properties of osteoblasts (OBs) isolated from the axial skeleton (tOBs) differ from OBs of the orofacial skeleton (mOBs) due to the different embryological origins of the bones. The aim of the study was to assess and compare the regenerative potential of allogenic bone marrow‐derived mesenchymal progenitor cells with allogenic tOBs and allogenic mOBs in combination with a mPCL–TCP scaffold in critical‐sized segmental bone defects in sheep tibiae. After 6 months, the tibiae were explanted and underwent biomechanical testing, micro‐computed tomography (microCT) and histological and immunohistochemical analyses. Allogenic MPCs demonstrated a trend towards a better outcome in biomechanical testing and the mean values of newly formed bone. Biomechanical, microCT and histological analysis showed no significant differences in the bone regeneration potential of tOBs and mOBs in our in vitro study, as well as in the bone regeneration potential of different cell types in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Multi‐modal imaging for assessment of tissue‐engineered bone in a critical‐sized calvarial defect mouse model

Tissue‐engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end‐point evaluations requiring the sacrifice of animals at specific time points. Due to differences in animal response to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non‐invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each animal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical‐sized calvarial defect mouse model. Osteogenesis of the TEB was monitored through signal variation, using magnetic resonance imaging (MRI) and near‐infrared (NIR) optical imaging with IRDye® 800CW BoneTag^TM (800CW BT, a bone‐specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the control (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a signal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi‐modal non‐invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright © 2015 John Wiley & Sons, Ltd.     

Gelatin‐ and hydroxyapatite‐based cryogels for bone tissue engineering: synthesis,characterization, in vitro and in vivo biocompatibility

The aim of this study was the synthesis and characterization of gelatin‐ and hydroxyapatite (osteoconductive component of bone)‐based cryogels for tissue‐engineering applications. Preliminary in vitro and in vivo biocompatibility tests were conducted. Gelatin‐ and hydroxyapatite‐based cryogels of varying concentrations were synthesized using glutaraldehyde as the crosslinking agent. Chemical structure, pore morphology, pore size distribution, mechanical properties, swelling characteristics and degradation profiles of the synthesized cryogels were demonstrated by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), mercury porosimetry, a mechanical test device, swelling ratio tests and weight loss measurements, respectively. In vitro cell viability and in vivo biocompatility tests were performed in order to show the performance of the cryogels in the biological environment. Changing the concentrations of gelatin, hydroxyapatite and crosslinker changed the chemical structure, pore size and pore size distribution of the cryogels, which in turn resulted in the ultimate behaviour (mechanical properties, swelling ratio, degradation profile). In vitro cell culture tests showed the viability of the cells. The cryogels did not show any cytotoxic effects on the cells. Clinical outcomes and the gross pathological results demonstrated that there was no necrosis noted in the abdominal and thoracic regions at the end of implantation and the implanted cryogel was found to be non‐irritant and non‐toxic at 12 weeks of implantation. Copyright © 2013 John Wiley & Sons, Ltd.     

Human tissue‐engineered bone produced in clinically relevant amounts using a semi‐automated perfusion bioreactor system: a preliminary study

The aim of this study was to evaluate a semi‐automated perfusion bioreactor system for the production of clinically relevant amounts of human tissue‐engineered bone. Human bone marrow stromal cells (hBMSCs) of eight donors were dynamically seeded and proliferated in a perfusion bioreactor system in clinically relevant volumes (10 cm³) of macroporous biphasic calcium phosphate scaffolds (BCP particles, 2–6 mm). Cell load and distribution were shown using methylene blue staining. MTT staining was used to demonstrate viability of the present cells. After 20 days of cultivation, the particles were covered with a homogeneous layer of viable cells. Online oxygen measurements confirmed the proliferation of hBMSCs in the bioreactor. After 20 days of cultivation, the hybrid constructs became interconnected and a dense layer of extracellular matrix was present, as visualized by scanning electron microscopy (SEM). Furthermore, the hBMSCs showed differentiation towards the osteogenic lineage as was indicated by collagen type I production and alkaline phosphatase (ALP) expression. We observed no significant differences in osteogenic gene expression profiles between static and dynamic conditions like ALP, BMP2, Id1, Id2, Smad6, collagen type I, osteocalcin, osteonectin and S100A4. For the donors that showed bone formation, dynamically cultured hybrid constructs showed the same amount of bone as the statically cultured hybrid constructs. Based on these results, we conclude that a semi‐automated perfusion bioreactor system is capable of producing clinically relevant and viable amounts of human tissue‐engineered bone that exhibit bone‐forming potential after implantation in nude mice. Copyright © 2009 John Wiley & Sons, Ltd.     

The effects of different vascular carrier patterns on the angiogenesis and osteogenesis of BMSC–TCP‐based tissue‐engineered bone in beagle dogs

Bone repair using tissue‐engineered bone (TEB) in a large defect or accompanied by a poor recipient vascular bed is a long‐standing challenge. Surgical vascular carrier patterns of vascular bundle (VB) and arteriovenous loop (AV loop) have been shown to improve the vascularization and repair capacity of TEB. However, the effects of these different vascular carrier patterns on angiogenesis and osteogenesis in TEB have never been evaluated. Here, TEB was constructed with bone marrow mesenchymal stem cells (BMSCs) and β‐TCP and prevascularized by the VB or AV loop technique in beagle dogs. The vascularization and bone formation in TEB were quantitatively compared using Microfil perfusion, histological examination and CT and micro‐CT analyses. The distribution and constitution of the neovasculature were analysed to determine the underlying mechanism of angiogenesis. The results showed that prevascularized TEB generated bone tissue faster and more homogeneously than untreated TEB. The VB technique was found to strike a better balance between bone regeneration and β‐TCP scaffold degradation than the AV loop strategy, which resulted in more vascularization but less bone yield, due to faster degradation of the β‐TCP scaffold. This study indicates that a suitable triangular relationship, composed of bone regeneration, scaffold degradation and vasculature, is critical to TEB construction. Copyright © 2015 John Wiley & Sons, Ltd.     

Engineering axially vascularized bone in the sheep arteriovenous‐loop model

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)‐loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein‐2 (rhBMP‐2), were harvested and directly autotransplanted in combination with β‐tricalcium phosphate–hydroxyapatite (β‐TCP–HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP‐2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β‐TCP–HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro‐computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV‐loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP‐2 stimulation in about 12 weeks in the sheep AV‐loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies. Copyright © 2012 John Wiley & Sons, Ltd.     

Bone tissue engineering in oral peri‐implant defects in preclinical in vivo research: A systematic review and meta‐analysis

The regeneration and establishment of osseointegration within oral peri‐implant bone defects remains a clinical challenge. Bone tissue engineering (BTE) is emerging as a promising alternative to autogenous and/or biomaterial‐based bone grafting. The objective of this systematic review was to answer the focused question: in animal models, do cell‐based BTE strategies enhance bone regeneration and/or implant osseointegration in experimental peri‐implant defects, compared with grafting with autogenous bone or only biomaterial scaffolds? Electronic databases were searched for controlled animal studies reporting on peri‐implant defects and implantation of mesenchymal stem cells (MSC) or other cells seeded on biomaterial scaffolds, following Preferred Reporting Items for Systematic reviews and Meta‐Analyses (PRISMA) guidelines. Random effects meta‐analyses were performed for the outcomes histomorphometric bone area fraction (BA) and bone‐to‐implant contact (BIC). Nineteen studies reporting on large animal models (dogs and sheep) were included. Experimental defects were created surgically (16 studies) or via ligature‐induced peri‐implantitis (LIPI, three studies). In general, studies presented with an unclear to high risk of bias. In most studies, MSC were used in combination with alloplastic mineral phase or polymer scaffolds; no study directly compared cell‐loaded scaffolds vs. autogenous bone. In three studies, cells were also modified by ex vivo gene transfer of osteoinductive factors. The meta‐analyses indicated statistically significant benefits in favour of: (a) cell‐loaded vs. cell‐free scaffolds [weighted mean differences (WMD) of 10.73–12.30% BA and 11.77–15.15% BIC] in canine surgical defect and LIPI models; and (b) gene‐modified vs. unmodified cells (WMD of 29.44% BA and 16.50% BIC) in canine LIPI models. Overall, heterogeneity in the meta‐analyses was high (I² 70–88%); considerable variation was observed among studies regarding the nature of cells and scaffolds used. In summary, bone regeneration and osseointegration in peri‐implant defects are enhanced by the addition of osteogenic cells to biomaterial scaffolds. Although the direction of treatment outcome is clearly in favour of BTE strategies, due to the limited magnitude of treatment effect observed, no conclusive statements regarding the clinical benefit of such procedures for oral indications can yet be made. Copyright © 2017 John Wiley & Sons, Ltd.     

Novel standardized massive bone defect model in rats employing an internal eight‐hole stainless steel plate for bone tissue engineering

Massive bone defects are a challenge in orthopaedic research. Defective regeneration leads to bone atrophy, non‐union of bone, and physical morbidity. Large animals are important models, however, production costs are high, nursing is complex, and evaluation methods are limited. A suitable laboratory animal model is required to explore the underlying molecular mechanism and cellular process of bone tissue engineering. We designed a stainless steel plate with 8 holes; the middle 2 holes were used as a guide to create a standardized critical size defect in the femur of anaesthetized rats. The plate was fixed to the bone using 6 screws, serving as an inner fixed bracket to secure a tricalcium phosphate implant seeded with green fluorescent protein‐positive rat bone marrow mesenchymal stem cells within the defect. In some animals, we also grafted a vessel bundle into the lateral side of the implant, to promote vascularized bone tissue engineering. X‐ray, microcomputed tomography, and histological analyses demonstrated the stainless steel plate resulted in a stable large segmental defect model in the rat femur. Vascularization significantly increased bone formation and implant degradation. Moreover, survival and expansion of green fluorescent protein‐positive seeded cells could be clearly monitored in vivo at 1, 4, and 8 weeks postoperation via fluorescent microscopy. This standardized large segmental defect model in a small animal may help to advance the study of bone tissue engineering. Furthermore, availability of antibodies and genetically modified rats could help to dissect the precise cellular and molecular mechanisms of bone repair.     

In vivo implantation of a tissue engineered stem cell seeded hemi‐laryngeal replacement maintains airway,phonation, and swallowing in pigs

Laryngeal functional impairment relating to swallowing, vocalisation, and respiration can be life changing and devastating for patients. A tissue engineering approach to regenerating vocal folds would represent a significant advantage over current clinical practice. Porcine hemi‐larynx were de‐cellularised under negative pressure. The resultant acellular scaffold was seeded with human bone marrow derived mesenchymal stem cells and primary human epithelial cells. Seeded scaffolds were implanted orthotopically into a defect created in the thyroid cartilage in 8 pigs and monitored in vivo for 2 months. In vivo assessments consisted of mucosal brushing and bronchoscopy at 1, 2, 4, and 8 weeks post implantation followed by histological evaluation post termination. The implanted graft had no adverse effect on respiratory function in 6 of the 8 pigs; none of the pigs had problems with swallowing or vocalisation. Six out of the 8 animals survived to the planned termination date; 2 animals were terminated due to mild stenosis and deep tissue abscess formation, respectively. Human epithelial cells from mucosal brushings could only be identified at Weeks 1 and 4. The explanted tissue showed complete epithelialisation of the mucosal surface and the development of rudimentary vocal folds. However, there was no evidence of cartilage remodelling at the relatively early censor point. Single stage partial laryngeal replacement is a safe surgical procedure. Replacement with a tissue engineered laryngeal graft as a single procedure is surgically feasible and results in appropriate mucosal coverage and rudimentary vocal fold development.     

Tubular open‐porous β‐tricalcium phosphate polycaprolactone scaffolds as guiding structure for segmental bone defect regeneration in a novel sheep model

Large segmental bone defect reconstruction with sufficient functional restoration is one of the most demanding challenges in orthopaedic surgery. Available regenerative treatment options, as the vascularized bone graft transfer, the Masquelet technique or the Ilizarov distraction osteogenesis, are associated with specific indications and distinct limitations. As an alternative, a hollow cylindrical ceramic‐polymer composite scaffold (β‐tricalcium phosphate and poly‐lactid co‐ε‐ caprolactone), facilitating a strong surface guiding effect for tissue ingrowth (group 1; n = 6) was investigated here. In combination with an additional autologous, cancellous bone graft filling, the scaffold's ability to work as an open‐porous membrane to improve the defect healing process was analysed (group 2; n = 6). A novel model of a critical size (40 mm) tibia osteotomy defect stabilized with an external hybrid‐ring fixator, was established in sheep. Segmental defect regeneration and tissue organization in relation to the scaffold were analysed radiologically, (immune‐) histologically, and with second‐harmonic generation imaging 12 weeks after surgery. The scaffold's tubular shape and open‐porous structure controlled the collagen fibre orientation within the bone defect and guided the following mineralization process along the scaffold surface. In combination with the osteoinductive stimulus, a unilateral bony bridging of the critically sized defect was achieved in one third of the animals. The external hybrid‐ring fixator was appropriate for large segmental defect stabilization in sheep.