Low molecular weight poly((d,l)-lactide-co-caprolactone) liquid inks for diluent-free DLP printing of cell culture platforms

Digital light processing (DLP) printing offers the possibility of fabricating complex objects in a fast and reproducible manner. A main requirement for DLP printing is the use of inks with low viscosities that can flow under the printing platform in a short period of time. Its exploitation in tissue engineering applications has been centered on the use of hydrogel forming materials diluted in aqueous solutions or the use of polyesters in combination with diluents and heating platforms that aid in the reduction of their viscosity. The use of diluents, however, modifies the mechanical properties and reduces the shape fidelity of the printed objects and, the use of heating platforms results in vats with heterogeneous temperatures and ink viscosities. Here, we report on the synthesis of a library of methacrylated low molecular weight (<3000 g mol−1) homopolymers ((P(D,L)LA and PCL) and copolymers (P((D,L)LA-co-CL)) of 2- and 3-arms based on (D,L)-lactide and ε-caprolactone. The resulting inks possessed low viscosity that made them printable in the absence of diluents and heating elements. DLP printing of cubical and cylindrical patterns resulted in objects with a higher shape fidelity than their counterparts fabricated using diluents and with printed features on the order of 300 μm. The printed materials were biocompatible and supported the growth of human mesenchymal stem cells (hMSCs). Moreover, the variations in the composition resulted in polymers that enabled the attachment of hMSCs to different extents, leading to the formation of well-adhered cell monolayers or loosely adhered cell aggregates.The authors acknowledge the funding bodies and support through the EMAKIKER grant. S. C.-E. acknowledges the Spanish Ministry of Science and Innovation (MICINN) – State Investigation Agency (AEI) (PID2020-114901RA-I00). S. C.-E. and S. R.-D. acknowledge the Basque Government (PIBA_2022_1_0006). G. L.-J. acknowledges the Basque Government Predoctoral grant PRE_2021_1_0403. S. C.-E. and L. I. acknowledge the Provincial Council of Guipuzcoa. The project that gave rise to these results received the support of a fellowship from the “laCaixa” Foundation (ID100010434). The fellowship code is 117145. S. C.-E. acknowledges funding from the University of the Basque Country UPV/EHU within the framework of Grupos de Investigación (GIU21/033). A. A. acknowledges funding from PID2021-127191OB-I00 and RTI2018-101708-A-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. Grant RYC2018-025502-I funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”

Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds

The extracellular matrix (ECM) is a complex network with multiple functions, including specific functions during tissue regeneration. Precisely, the properties of the ECM have been thoroughly used in tissue engineering and regenerative medicine research, aiming to restore the function of damaged or dysfunctional tissues. Tissue decellularization is gaining momentum as a technique to obtain potentially implantable decellularized extracellular matrix (dECM) with well-preserved key components. Interestingly, the tissue-specific dECM is becoming a feasible option to carry out regenerative medicine research, with multiple advantages compared to other approaches. This review provides an overview of the most common methods used to obtain the dECM and summarizes the strategies adopted to decellularize specific tissues, aiming to provide a helpful guide for future research development.This work was supported by the Spanish “Programa Estatal de I+D+i Orientada a los Retos de la Sociedad”, grant number RTI2018-101708-A-I00. S.R.-C. was supported by Fomento de San Sebastian innovative talent programme, grant number 0508/2019/0009. R.R.-H. was supported by Spanish State Training Subprogramme, grant number PRE2018-084542. A.A. was supported by Spanish State Subprogramme of Incorporation Ramón y Cajal, grant number RYC2018-025502-I, and 2019 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation, grant number IN[19]_CMA_BIO_0119

Clonal dynamics in osteosarcoma defined by RGB marking

Osteosarcoma is a type of bone tumour characterized by considerable levels of phenotypic heterogeneity, aneuploidy, and a high mutational rate. The life expectancy of osteosarcoma patients has not changed during the last three decades and thus much remains to be learned about the disease biology. Here, we employ a RGB-based single-cell tracking system to study the clonal dynamics occurring in a de novo-induced murine osteosarcoma model. We show that osteosarcoma cells present initial polyclonal dynamics, followed by clonal dominance associated with adaptation to the microenvironment. Interestingly, the dominant clones are composed of subclones with a similar tumour generation potential when they are re-implanted in mice. Moreover, individual spontaneous metastases are clonal or oligoclonal, but they have a different cellular origin than the dominant clones present in primary tumours. In summary, we present evidence that osteosarcomagenesis can follow a neutral evolution model, in which different cancer clones coexist and propagate simultaneously.We thank ISCIII and CNIO flow cytometry and cell sorting units for their participation in our studies. We are thankful to the CCEH-Fred Hutchinson Cancer Research Center for LAM-PCR service. We acknowledge Raquel Pérez Tavarez, María Blázquez Mesa, Alicia Giménez Sánchez, Elena Calvo Cazalilla, and Monserrat Arroyo Correas for useful help on the pathology studies; and Teresa Cejalvo, Isabel Cubillo Moreno, and Miguel Angel Rodríguez-Milla for their contributions in experimental setup. We thank the visual artist Isabella Lacquaniti for her help with drawings and schematics. We are also thankful to the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 and PI14CIII/00005 to J.G.-C., FIS: CP11/00206 to A.A., and RTICC: RD12/0036/0027 to J.G.-C.), the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C.), the Asociación Pablo Ugarte, and the Asociación Afanion for grants support.S

A Humanized Bone Niche Model Reveals Bone Tissue Preservation Upon Targeting Mitochondrial Complex I in Pseudo-Orthotopic Osteosarcoma

A cogent issue in cancer research is how to account for the effects of tumor microenvironment (TME) on the response to therapy, warranting the need to adopt adequate in vitro and in vivo models. This is particularly relevant in the development of strategies targeting cancer metabolism, as they will inevitably have systemic effects. For example, inhibition of mitochondrial complex I (CI), despite showing promising results as an anticancer approach, triggers TME-mediated survival mechanisms in subcutaneous osteosarcoma xenografts, a response that may vary according to whether the tumors are induced via subcutaneous injection or by intrabone orthotopic transplantation. Thus, with the aim to characterize the TME of CI-deficient tumors in a model that more faithfully represents osteosarcoma development, we set up a humanized bone niche ectopic graft. A prominent involvement of TME was revealed in CI-deficient tumors, characterized by the abundance of cancer associated fibroblasts, tumor associated macrophages and preservation of osteocytes and osteoblasts in the mineralized bone matrix. The pseudo-orthotopic approach allowed investigation of osteosarcoma progression in a bone-like microenvironment setting, without being invasive as the intrabone cell transplantation. Additionally, establishing osteosarcomas in a humanized bone niche model identified a peculiar association between targeting CI and bone tissue preservation

Adipose-derived stem cells and platelet-rich plasma for preventive treatment of bisphosphonate-related osteonecrosis of the jaw in a murine model

This is the author’s version of a work that was accepted for publication in Journal of Cranio-Maxillofacial Surgery. A definitive version was subsequently published in Journal of Cranio-Maxillofacial Surgery 43.7 (2015) DOI: 10.1016/j.jcms.2015.04.026Objectives: The main challenge in treating bisphosphonate-related osteonecrosis of the jaw (BRONJ) is the absence of an effective established treatment. We aimed to compare different potentially preventive treatments for BRONJ after dental extractions in zoledronic acid (ZA)-treated animals. We studied the local application of different combinations of adipose-derived stem cells (ASCs) with or without previous stimulation with bone morphogenetic protein 2 (BMP-2) and platelet-rich plasma (PRP) in rats. Material and methods: Fifty-six male Wistar rats were treated with ZA for 9 weeks. Dental extractions were performed in the eighth week, and the animals were divided into 4 groups. In group 1 (n ¼ 14), alveolar coverage with mucoperiosteal flap was performed. In group 2 (n ¼ 14), PRP was applied over the sockets and covered with the flap. In group 3 (n ¼ 15), allogeneic ASCs with PRP were applied and covered with the flap. In group 4 (n ¼ 13), animals were treated with ASCs cultured with BMP-2, PRP, and flap coverage. Histologic, fluorescence, and radiologic studies of the maxillae were performed. Results: ASC-treated animals showed lower frequency of osteonecrosis (14% vs 50%, p ¼ 0.007) and greater bone turnover (p ¼ 0.024) and osteoclast count (p ¼ 0.045) than those not receiving the ASC treatment. Conclusions: In this high-risk model, ASC-based treatments seem to prevent BRONJ more effectively than mucosal flap with or without PRP. The combination of ASCs and PRP appears to be synergistic, and the addition of BMP-2 could further improve the resultsThis study was supported by a grant from the Spanish Ministry of Health and Consumer Affairs (FIS PI10/01991) and a RETICS grant from ISCIII (RD12/0019/0035) and the Education Council of Madrid (P2010/BMD-2420)

Osteosarcoma: Cells-of-Origin, Cancer stem cells, and targeted therapies

Osteosarcoma (OS) is the most common type of primary solid tumor that develops in bone. Although standard chemotherapy has significantly improved long-term survival over the past few decades, the outcome for those patients with metastatic or recurrent OS remains dismally poor and, therefore, novel agents and treatment regimens are urgently required. A hypothesis to explain the resistance of OS to chemotherapy is the existence of drug resistant CSCs with progenitor properties that are responsible of tumor relapses and metastasis. These subpopulations of CSCs commonly emerge during tumor evolution from the cell-of-origin, which are the normal cells that acquire the first cancer-promotingmutations to initiate tumor formation. In OS, several cell types along the osteogenic lineage have been proposed as cell-of-origin. Both the cell-of-origin and their derived CSC subpopulations are highly influenced by environmental and epigenetic factors and, therefore, targeting the OS-CSC environment and niche is the rationale for many recently postulated therapies. Likewise, some strategies for targeting CSC-associated signaling pathways have already been tested in both preclinical and clinical settings. This review recapitulates current OS cell-of-origin models, the properties of the OS-CSC and its niche, and potential new therapies able to target OS-CSCs

Decellularization of xenografted tumors provides cell-specific in vitro 3D environment

In vitro cell culture studies are common in the cancer research field, and reliable biomimetic 3D models are needed to ensure physiological relevance. In this manuscript, we hypothesized that decellularized xenograft tumors can serve as an optimal 3D substrate to generate a top-down approach for in vitro tumor modeling. Multiple tumor cell lines were xenografted and the formed solid tumors were recovered for their decellularization by several techniques and further characterization by histology and proteomics techniques. Selected decellularized tumor xenograft samples were seeded with the HCC1806 human triple-negative breast cancer (TNBC) basal-like subtype cell line, and cell behavior was compared among them and with other control 2D and 3D cell culture methods. A soft treatment using Freeze-EDTA-DNAse allows proper decellularization of xenografted tumor samples. Interestingly, proteomic data show that samples decellularized from TNBC basal-like subtype xenograft models had different extracellular matrix (ECM) compositions compared to the rest of the xenograft tumors tested. The in vitro recellularization of decellularized ECM (dECM) yields tumor-type–specific cell behavior in the TNBC context. Data show that dECM derived from xenograft tumors is a feasible substrate for reseeding purposes, thereby promoting tumor-type–specific cell behavior. These data serve as a proof-of-concept for further potential generation of patient-specific in vitro research models.Grant RTI2018-101708-A-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe. Grants RYC2018-025502-I and PRE2018-084542 are funded by MCIN/AEI/10.13039/501100011033 and by ESF Investing in your future. Grant MDM-2017-0720 Maria de Maeztu Units of Excellence Program funded by the Spanish State Research Agency. Grant KK-2019/00093 Elkartek program funded by Basque Government. Grant CICBMG_PhD_03_2021 funded by CICbiomaGUNE and Polymat. Grant CICBMG_PhD_05_2019 funded by CICbiomaGUNE and Polymat. 2019 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation, grant number IN[19]_CMA_BIO_0119. The BBVA Foundation accepts no responsibility for the opinions, statements, and contents included, which are entirely the responsibility of the authors

Biological Properties of Solid Free Form Designed Ceramic Scaffolds with BMP-2: In Vitro and In Vivo Evaluation

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures

c-Fos induces chondrogenic tumor formation in immortalized human mesenchymal progenitor cells

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation.This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the “Miguel Servet” program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees.S

Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches