An innovative 3D hydroxyapatite patient-specific implant for maxillofacial bone reconstruction: A case series of 13 patients.

The study aimed to evaluate and discuss the use of an innovative PSI made of porous hydroxyapatite, with interconnected porosity promoting osteointegration, called MyBone Custom® implant (MBCI), for maxillofacial bone reconstruction. A multicentric cohort of 13 patients underwent maxillofacial bone reconstruction surgery using MBCIs for various applications, from genioplasty to orbital floor reconstruction, including zygomatic and mandibular bone reconstruction, both for segmental defects and bone augmentation. The mean follow-up period was 9 months (1-22 months). No infections, displacements, or postoperative fractures were reported. Perioperative modifications of the MBCIs were possible when necessary. Additionally, surgeons reported significant time saved during surgery. For patients with postoperative CT scans, osteointegration signs were visible at the 6-month postoperative follow-up control, and continuous osteointegration was observed after 1 year. The advantages and disadvantages compared with current techniques used are discussed. MBCIs offer new bone reconstruction possibilities with long-term perspectives, while precluding the drawbacks of titanium and PEEK. The low level of postoperative complications associated with the high osteointegration potential of MBCIs paves the way to more extensive use of this new hydroxyapatite PSI in maxillofacial bone reconstruction.

How Does Customized Cutting Guide Design Affect Accuracy and Ergonomics in Pelvic Tumor Resection? A Study in Cadavers.

BACKGROUND: Customized cutting guides are technical aids that make primary pelvic bone tumor resection safer and more reliable. Although the effectiveness of such devices appears to be widely accepted, their conception and design remain varied. Two main designs have been reported: the heavier block-type customized cutting guides and the lighter patch-type customized cutting guides. As recent tools, there must be more evidence regarding the impact of design on their accuracy and ergonomics. Thus, an evaluation of their respective performances appears warranted. QUESTIONS/PURPOSES: In a cadaver model, we assessed whether (1) a thinner, patch-type customized cutting guide design results in resections that are closer to the planned resections than the heavier block-type customized cutting guides, and (2) the patch-type customized cutting guide design is more ergonomic than the block-type customized cutting guide with improved usability in surgery (in terms of bulkiness, ease of placement, primary and secondary stability, and stability during cutting). METHODS: We conducted an experimental study involving five fresh whole-body anatomic specimens (three women and two men with a median age of 79 years and median weight of 66 kg) by simulating six virtual tumors in three areas according to the Enneking classification (Zones I: iliac wing, II: periacetabular area, and I and IV: sacroiliac joint area). We compared the impact of the customized cutting guide's design on performance in terms of the resection margin accuracy using CT scan analysis (deviation from the planned margin at the closest point and the maximum deviation from the planned margin) and the intraoperative ergonomic score under conditions simulating those of an oncologic resection of a bone tumor (with a range of 0 to 100, with 100 being best). RESULTS: The patch customized cutting guides performed slightly better than the block customized cutting guides regarding deviation from the planned margin at the closest point, with median values of 1 mm versus 2 mm (difference of medians 1 mm; p = 0.02) and maximum deviation from the planned margin of 3 versus 4 mm (difference of medians 1 mm; p = 0.002). In addition, the patch design was perceived to be slightly more ergonomic than the block design, with a 92% median score versus 84% for the block design (difference of medians 8%; p = 0.03). CONCLUSION: We observed an equivalence in performance regarding accuracy and ergonomics, with slight advantages for patch customized cutting guides, especially in complex zones (Zone I and IV). Owing to a small cohort in a cadaver study, these results need independent replication. CLINICAL RELEVANCE: The patch-type customized cutting guide with thinner contact spots to the bone in specific areas and less soft tissue dissection might offer an advantage over a larger block design for achieving negative oncologic bony margins, but it does not address issues of soft tissue margins.

A Simple 3D Cell Culture Method for Studying the Interactions between Human Mesenchymal Stromal/Stem Cells and Patients Derived Glioblastoma.

We have developed a 3D biosphere model using patient-derived cells (PDCs) from glioblastoma (GBM), the major form of primary brain tumors in adult, plus cancer-activated fibroblasts (CAFs), obtained by culturing mesenchymal stem cells with GBM conditioned media. The effect of MSC/CAFs on the proliferation, cell-cell interactions, and response to treatment of PDCs was evaluated. Proliferation in the presence of CAFs was statistically lower but the spheroids formed within the 3D-biosphere were larger. A treatment for 5 days with Temozolomide (TMZ) and irradiation, the standard therapy for GBM, had a marked effect on cell number in monocultures compared to co-cultures and influenced cancer stem cells composition, similar to that observed in GBM patients. Mathematical analyses of spheroids growth and morphology confirm the similarity with GBM patients. We, thus, provide a simple and reproducible method to obtain 3D cultures from patient-derived biopsies and co-cultures with MSC with a near 100% success. This method provides the basis for relevant in vitro functional models for a better comprehension of the role of tumor microenvironment and, for precision and/or personalized medicine, potentially to predict the response to treatments for each GBM patient.

Digital chain for pelvic tumor resection with 3D-printed surgical cutting guides.

Surgical cutting guides are 3D-printed customized tools that help surgeons during complex surgeries. However, there does not seem to be any set methodology for designing these patient-specific instruments. Recent publications using pelvic surgical guides showed various designs with no clearly classified or standardized features. We, thus, developed a systematic digital chain for processing multimodal medical images (CT and MRI), designing customized surgical cutting guides, and manufacturing them using additive manufacturing. The aim of this study is to describe the steps in the conception of surgical cutting guides used in complex oncological bone tumor pelvic resection. We also analyzed the duration of the surgical cutting guide process and tested its ergonomics and usability with orthopedic surgeons using Sawbones models on simulated tumors. The original digital chain made possible a repeatable design of customized tools in short times. Preliminary testing on synthetic bones showed satisfactory results in terms of design usability. The four artificial tumors (Enneking I, Enneking II, Enneking III, and Enneking I+IV) were successfully resected from the Sawbones model using this digital chain with satisfactory ergonomic outcomes. This work validates a new digital chain conception and production of surgical cutting guides. Further works with quantitative margin assessments on anatomical subjects are needed to better assess the design implications of patient-specific surgical cutting guide instruments in pelvic tumor resections.

Signature of the vascular tumor microenvironment as a marker of the therapeutic response to doxorubicin in a preclinical model of osteosarcoma.

Predicting a response of osteosarcoma patients to chemotherapy, such as doxorubicin or high-dose methotrexate cocktail, remains a challenge in the clinic. Moreover, the prognostic value of currently used necrosis analysis is debatable. New markers of the therapeutic response or the prognostic response are urgently needed. The microenvironment plays a key role in the vascularization of highly heterogeneous tumors. Using the syngeneic MOS-J mouse model of osteosarcoma, we focused our study on the immunohistochemistry of tumor vascularization in order to identify new vessel markers, and to search for potential markers of the therapeutic response. Endomucin+, CD31+, and α-SMA+-positive elements were quantified in control (n=6) and doxorubicin-treated (n=6) mice in three different intra-tumor locations. We also used co-labeling to assess CD31+/Endomucin+ and CD31+/α-SMA+ co-expression. We identified a central tumor zone with a low vascularization profile for all of these markers. We identified two distinct types of vessels: CD31+/Endomucin+ vessels with a sprouting, neo-angiogenic, interlaced appearance, and CD31+/α-SMA+ vessel with a well-defined, mature structure. Doxorubicin appeared to reduce CD31+ expression in the tumor invasion front. In the doxorubicin-sensitive model, there were four times more CD31+/α-SMA+ elements than in the poorly responsive model. Therefore, we propose a methodology based on immunohistochemistry and multiplexed immunofluorescence to use endomucin as a promising new vascular marker in the osteosarcoma model. Moreover, our results suggest that CD31+/α-SMA+ vessels could be considered to be indicators of vasculature normalization and they may be used as specific markers of a good therapeutic response.

A Preliminary Study for an Intraoperative 3D Bioprinting Treatment of Severe Burn Injuries.

Intraoperative three-dimensional fabrication of living tissues could be the next biomedical revolution in patient treatment. APPROACH: We developed a surgery-ready robotic three-dimensional bioprinter and demonstrated that a bioprinting procedure using medical grade hydrogel could be performed using a 6-axis robotic arm in vivo for treating burn injuries. RESULTS: We conducted a pilot swine animal study on a deep third-degree severe burn model. We observed that the use of cell-laden bioink as treatment substantially affects skin regeneration, producing in situ fibroblast growth factor and vascular endothelial growth factor, necessary for tissue regeneration and re-epidermalization of the wound. CONCLUSIONS: We described an animal study of intraoperative three-dimensional bioprinting living tissue. This emerging technology brings the first proof of in vivo skin printing feasibility using a surgery-ready robotic arm-based bioprinter. Our positive outcome in skin regeneration, joined with this procedure's feasibility, allow us to envision the possibility of using this innovative approach in a human clinical trial in the near future.

Chondrogenic and BMP-4 primings confer osteogenesis potential to human cord blood mesenchymal stromal cells delivered with biphasic calcium phosphate ceramics.

Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.

In situ production of pre-vascularized synthetic bone grafts for regenerating critical-sized defects in rabbits.

Reconstructing large bone defects caused by severe trauma or resection of tumors remains a challenge for surgeons. A fibula free flap and its vascularized bed can be transplanted to the reconstruction site to achieve healing. However, this technique adds morbidity, and requires microsurgery and sculpting of the bone tissue to adapt the graft to both the vasculature and the anatomy of the defect. The aim of the current study was to evaluate an alternative approach consisting of the in situ production of a pre-vascularized synthetic bone graft and its subsequent transplantation to a critical-sized bone defect. 3D printed chambers containing biphasic calcium phosphate (BCP) granules, perfused by a local vascular pedicle, with or without the addition of stromal vascular fraction (SVF), were subcutaneously implanted into New Zealand White female rabbits. SVF was prepared extemporaneously from autologous adipose tissue, the vascular pedicle was isolated from the inguinal site, while BCP granules alone served as a control group. After 8 weeks, the constructs containing a vascular pedicle exhibited abundant neovascularization with blood vessels sprouting from the pedicle, leading to significantly increased vascularization compared to BCP controls. Pre-vascularized synthetic bone grafts were then transplanted into 15 mm critical-sized segmental ulnar defects for a further 8 weeks. Micro-CT and decalcified histology revealed that pre-vascularization of synthetic bone grafts led to enhanced bone regeneration. This pre-clinical study demonstrates the feasibility and efficacy of the in situ production of pre-vascularized synthetic bone grafts for regenerating large bone defects, thereby addressing an important clinical need. STATEMENT OF SIGNIFICANCE: The current gold standard in large bone defect regeneration is vascularized fibula grafting. An alternative approach consisting of in situ production of a pre-vascularized synthetic bone graft and its subsequent transplantation to a bone defect is presented here. 3D printed chambers were filled with biphasic calcium phosphate granules, supplemented with autologous stromal vascular fraction and an axial vascular pedicle and subcutaneously implanted in inguinal sites. These pre-vascularized synthetic grafts were then transplanted into critical-sized segmental ulnar defects. Micro-CT and decalcified histology revealed that the pre-vascularized synthetic bone grafts led to higher bone regeneration than non-vascularized constructs. An alternative to vascularized fibula grafting is provided and may address an important clinical need for large bone defect reconstruction.

Reconstruction of Large Skeletal Defects: Current Clinical Therapeutic Strategies and Future Directions Using 3D Printing.

The healing of bone fractures is a well-orchestrated physiological process involving multiple cell types and signaling molecules interacting at the fracture site to replace and repair bone tissue without scar formation. However, when the lesion is too large, normal healing is compromised. These so-called non-union bone fractures, mostly arising due to trauma, tumor resection or disease, represent a major therapeutic challenge for orthopedic and reconstructive surgeons. In this review, we firstly present the current commonly employed surgical strategies comprising auto-, allo-, and xenograft transplantations, as well as synthetic biomaterials. Further to this, we discuss the multiple factors influencing the effectiveness of the reconstructive therapy. One essential parameter is adequate vascularization that ensures the vitality of the bone grafts thereby supporting the regeneration process, however deficient vascularization presents a frequently encountered problem in current management strategies. To address this challenge, vascularized bone grafts, including free or pedicled fibula flaps, or in situ approaches using the Masquelet induced membrane, or the patient's body as a bioreactor, comprise feasible alternatives. Finally, we highlight future directions and novel strategies such as 3D printing and bioprinting which could overcome some of the current challenges in the field of bone defect reconstruction, with the benefit of fabricating personalized and vascularized scaffolds.

Epinephrine Infiltration of Adipose Tissue Impacts MCF7 Breast Cancer Cells and Total Lipid Content.

BACKGROUND: Considering the positive or negative potential effects of adipocytes, depending on their lipid composition, on breast tumor progression, it is important to evaluate whether adipose tissue (AT) harvesting procedures, including epinephrine infiltration, may influence breast cancer progression. METHODS: Culture medium conditioned with epinephrine-infiltrated adipose tissue was tested on human Michigan Cancer Foundation-7 (MCF7) breast cancer cells, cultured in monolayer or in oncospheres. Lipid composition was evaluated depending on epinephrine-infiltration for five patients. Epinephrine-infiltrated adipose tissue (EI-AT) or corresponding conditioned medium (EI-CM) were injected into orthotopic breast carcinoma induced in athymic mouse. RESULTS: EI-CM significantly increased the proliferation rate of MCF7 cells Moreover EI-CM induced an output of the quiescent state of MCF7 cells, but it could be either an activator or inhibitor of the epithelial mesenchymal transition as indicated by gene expression changes. EI-CM presented a significantly higher lipid total weight compared with the conditioned medium obtained from non-infiltrated-AT of paired-patients. In vivo, neither the EI-CM or EI-AT injection significantly promoted MCF7-induced tumor growth. CONCLUSIONS: Even though conditioned media are widely used to mimic the secretome of cells or tissues, they may produce different effects on tumor progression, which may explain some of the discrepancy observed between in vitro, preclinical and clinical data using AT samples.

Should platelet-rich plasma be activated in fat grafts? An animal study.

BACKGROUND: The adjunction of platelet-rich plasma with graft fat has been the subject of a few clinical trials which have demonstrated its value in adipocyte survival. The aim of this study was to assess the different efficacies between activated and non-activated PRP on adipose cells in vitro and for adipose tissue graft survival in vivo. METHODS: The in vitro study assessed the effects of PRP on both the proliferation and adipocyte differentiation of adipose cells. For the in vivo study, 8 nude rats received 3 human fat injections as follows: 0.8 mL of fat + 0.2 mL of normal saline; 0.8 mL of fat + 0.2 mL of non-activated PRP; and 0.8 mL of fat + 0.2 mL of PRP activated with calcium chloride (CaCl2). The quantitative assessment of adipocyte survival was implemented after 3 months using histomorphometric analysis. Histological and immunohistochemical analysis were also performed to evaluate angiogenesis, inflammation and quality of adipocytes in the grafted tissue. RESULTS: We showed that activated PRP stimulated, in vitro, proliferation and differentiation of adipose cells. In vivo experiments indicated that CaCl2-activated PRP was more efficient than non-activated to prolong the survival of fat grafts in nude rats. The mean percentage areas occupied by viable adipocytes in the PRP-free group, non-activated PRP group and activated PRP group were 13%, 14% and 24% (p = 0.05%), respectively. Histological and immunohistochemical analysis revealed protective effect of activated PRP on inflammation and adipocyte death. CONCLUSION: This study showed that activation by CaCl2 improves the beneficial effects of PRP for fat graft maintenance.

Bone microenvironment has an influence on the histological response of osteosarcoma to chemotherapy: retrospective analysis and preclinical modeling.

Osteosarcoma, the most common malignant primary bone tumor, is currently treated with chemotherapy and surgery. The effectiveness of chemotherapy is evaluated by means of histological analysis of tumor necrosis, known as "the Huvos score". However, 25% of the patients initially considered good responders will relapse. In our practice, strong tissue heterogeneity around the residual viable cells of the osteosarcoma is observed, but this is not taken into account by the Huvos score, as it is only an average. The objective is to determine whether heterogeneity in the osteosarcoma's microenvironment can play a role in the histological response to chemotherapy. Two complementary approaches have been developed: (i) the therapeutic response to several monotherapies (ifosfamide, cisplatin, doxorubicin) has been compared to tumor growth and the necrosis levels in different preclinical syngeneic osteosarcoma models, mimicking various microenvironments by injecting the tumor cells into subcutaneous, intra-muscular paratibial, or intra-osseous sites; (ii) a retrospective analysis was performed on patients' osteoblastic osteosarcoma biopsies. Tissue localization mapping of residual live tumor cell colonies was evaluated for potential correlation with overall survival. The results of the preclinical studies showed a difference in tumor growth depending on the osteosarcoma model, with a higher rate in bone sites compared to subcutaneous tumors. For the therapeutic response, a higher response to doxorubicin was observed in the intra-osseous model compared to the intra-muscular model for tumor growth (P = 0.013) and necrosis (P = 0.007). These data strongly suggest that the microenvironment plays a role in how osteosarcoma responds to chemotherapy. The retrospective analysis showed no significant survival difference between residual cell sites, although the soft tissues may be seen as a potential negative factor.

Primary Fat Grafting to the Pectoralis Muscle during Latissimus Dorsi Breast Reconstruction.

BACKGROUND: Latissimus dorsi flap is one of the best options for immediate and delayed breast reconstruction. However, this technique is limited by the tissue volume provided by the flap. To improve breast volume while reducing complications, fat grafting is now very often used in addition to latissimus dorsi flap. To the best of our knowledge, fat grafting was always performed as a second-line surgery, at least a few months after the flap procedure. We aimed to report our experience with an associated breast reconstruction technique combining musculocutaneous latissimus dorsi flap with intrapectoral lipofilling for totally autologous breast reconstruction. METHODS: Between September 2014 and January 2015, 20 patients underwent this technique for unilateral autologous breast reconstruction (14 delayed and 6 immediate breast reconstructions). A mean harvested fat volume of 278 ml (range: 190-350 ml) and a mean injected fat volume of 228 ml (range: 170-280 ml) were used. RESULTS: None of the patients experienced complications, such as flap necrosis, breast skin necrosis, hematomas, or infection. One of the patients developed a seroma, which was treated with 3 drainage punctions. Only 2 patients underwent delayed fat grafting procedure. CONCLUSION: Totally autologous breast reconstruction combining latissimus dorsi flap and intrapectoral fat grafting in the same procedure is a new technique allowing increased breast volume in a single surgery.

Adipocyte differentiation influences the proliferation and migration of normal and tumoral breast epithelial cells.

Stromal tissue regulates the development and differentiation of breast epithelial cells, with adipocytes being the main stromal cell type. The aim of the present study was to evaluate the effect of adipocyte differentiation on proliferation and migration, as well as to assess the activity of heparanase and metalloproteinase-9 (MMP-9), in normal (NMuMG) and tumoral (LM3) murine breast epithelial cells. NMuMG and LM3 cells were grown on irradiated 3T3-L1 cells (stromal support, SS) at various degrees of differentiation [preadipocytes (preA), poorly differentiated adipocytes (pDA) and mature adipocytes (MA)] and/or were incubated in the presence of conditioned medium (CM) derived from each of these three types of differentiated cells. Cells grown on a plastic support or in fresh medium served as the controls. Cell proliferation was measured with a commercial colorimetric kit, and the motility of the epithelial cells was evaluated by means of a wound-healing assay. Heparanase activity was assessed by quantifying heparin degradation, and the expression of MMP-9 was determined using Western blotting. The results indicate that cell proliferation was increased after 24 and 48 h in the NMuMG and LM3 cells grown on preA, pDA and MA SS. In the NMuMG cells cultured on SS in the presence of all three types of CM, proliferation was enhanced. LM3 cell migration was increased in the presence of all three types of CM and in cells grown on preA SS. Heparanase activity was increased in the NMuMG cells incubated with all three types of CM, and in the LM3 cells incubated with the CM from pDA and MA. Both the NMuMG and LM3 cell lines presented basal expression of MMP-9; however, a significant increase in MMP-9 expression was observed in the LM3 cells incubated with each of the three types of CM. In conclusion, adipocyte differentiation influences normal and tumoral breast epithelial cell proliferation and migration. Heparanase and MMP-9 appear to be involved in this regulation. The experimental model presented in this study is in keeping with the characteristics of the physiological environment of breast epithelial cells, in terms of both the soluble and insoluble factors present and the stromal structure per se.