The human amniotic membrane (hAM), derived from the placenta, possesses a low (nay inexistant) immunogenicity and exerts an anti-inflammatory, anti-fibrotic, antimicrobial, antiviral and analgesic effect. It is a source of stem cells and growth factors promoting tissue regeneration. hAM acts as an anatomical barrier with adequate mechanical properties (permeability, stability, elasticity, flexibility, resorbability) preventing the proliferation of fibrous tissue and promoting early neovascularization of the surgical site. Cryopreservation and lyophilization, with sometimes additional decellularization process, are the main preservation methods for hAM storage.

We examined the use of hAM in orthopaedic and maxillofacial bone surgery, specially to shorten the induced membrane technique (Gindraux, 2017). We investigated the cell survival in cryopreserved hAM (Laurent, 2014) and the capacity of intact hAM of in vitro osteodifferentiation (Gualdi, 2019). We explored its in vivo osteogenic potential in an ectopic model (Laurent, 2017) and, with Inserm U1026 BioTis, in a calvarial defect (Fenelon, 2018). Still piloted by U1026, decellularization and/or lyophilization process were developed (Fenelon, 2019) and, processed hAM capacities was assessed for guided bone regeneration (Fenelon 2020) and induced membrane technique (Fenelon, 2021) in mice.

We reported a limited function of hAM for bone defect management. In this light, we recognized medication-related osteonecrosis of the jaw (MRONJ) as appropriate model of disease to evaluate hAM impact on both oral mucosa and bone healing. We treated height compassionate patients (stage II, III) with cryopreserved hAM. A multicentric randomized clinical study (PHRC-I 2020 funding) will be soon conducted in France (regulatory and ethical authorization in progress).

The human amniotic membrane (hAM) may be helpful as a support for bone regeneration. To assess its potential for bone repair, a wide heterogeneity of preservation methods of hAM has been studied. The objectives of this study were: i) to assess bone regeneration potential of fresh versus cryopreserved hAM, and ii) to characterize hAM depending on four preservation methods. hAM was used either fresh (F-hAM), cryopreserved (C-hAM), lyophilized (L-hAM) or decellularized and lyophilized (DL-hAM). First, critical calvarial bone defects were performed in mice. Defects remained empty or were covered by F-hAM or C-hAM. Then, the cytotoxicity of the four preservation methods of hAM was assessed in vitro on human bone marrow mesenchymal stem cells (hBMSCs), and, their biocompatibility was evaluated in vivo in a rat subcutaneous model. X-Rays analysis showed that no calvarial defect was regenerated ad integrum. Bone regeneration was slightly enhanced by C-hAM. In vitro, the decellularization and the lyophilization process did not confer any cytotoxicity of the tissue compared to other preservation methods. In vivo, L-hAM and DL-hAM were easier to handle. Histological analysis of explanted samples from the rat indicated a slight to moderate inflammatory reaction with hAM. One month after surgery, a complete resorption of F-hAM and C-hAM implants occured, whereas L-hAM and DL-hAM were still observed. C-hAM has a limited potential for GBR. L-hAM and DL-hAM are biocompatible without cytotoxic effects. These preservation methods should be suitable in the field of bone regeneration.

Previous clinical studies have shown the efficacy of a foreign body-induced membrane combined with bone autograft for the reconstruction of traumatologic or pathologic large bone defects or, bone non union. This membrane, rich in mesenchymal stromal cells (MSC), avoids bone autograft resorption and promotes consolidation by revascularisation of the bone and secretion of growth factors. Reconstruction requires two different surgical stages: firstly, insertion of a cement spacer in the defect, and secondly, removal of the spacer, preservation of the foreign body-induced membrane and filling of the cavity by bone autograft. The optimal time to perform the second surgical stage remains unclear.

So, we aimed to correlate bone healing and, phenotype and function of cells isolated from the induced membrane, in patients whose second surgery was performed on average after 6 months (i.e. beyond the recommended time of one month). Cell phenotype was determined by flow cytometry and cell function by: alkaline Phosphatase enzyme activity, secretion of calcium and von Kossa staining. Second, using histological and immunohistochemistry studies, we aimed to determine the nature and function of induced membrane over time. Seven patients were included with their consent.

Results showed Treated patients achieved in all cases bone union (except for one patient) and in in vitro and histology and immunohistochemistry gave some indications which need to be completed in the future. First, patient age seemed to be an indicator of bone union speed and recurrent infection, appeared to influence in vitro MSC osteogenic potential and induced membrane structure. Second, we reported, in bone repair situation, the commitment over time in osteogenic lineage of a surprising multipotent tissue (induced membrane) able of vascularisation/ osteogenesis/ chondrogenesis at a precocious time. Finally, best time to perform the second stage (one month) could be easily exceeded since bone union occurred even at very late times.

In the area of 3D printing, more and more maxillofacial surgery departments are equipped with 3D printers to build their own anatomical models or surgical guides. Prior to be printable, the patients' DICOM imaging data has to be converted to a 3D virtual model, a 3D mesh. The file format most commonly used is the STL (Standard Tesselation Language) file format. Many programs exist that are able to convert DICOM data to STL files. Commercial software, such as Surgicase CMF© are FDA- and CE-approved whereas free programs, available online do not have the approval. However, the latter are often used anyway because of financial reasons. In this article, we investigate whether 6 of these software solutions are equivalent or not.

Thin slice CT imaging data of a patient's mandible (in DICOM file format) was converted to STL meshes with 6 different software solutions. One commercial program, Surgicase CMF©, was used to build the reference model. Then 5 free programs were used to create 5 models of the same mandible, specifying the same thresholding parameters: InVesalius 3.0, 3DimViewer 2.2.4, 3D Slicer, itk-Snap and Seg3D. All of these models were loaded in Netfabb Basic 6.4 to retrieve dimensional data, geometric information and the number of holes in each mesh. Finally, the models were then compared to the reference model using CloudCompare 2.6.2.

All models created with free software differed from the reference model in the 3 dimensions. Mean length difference was −0.74 mm [−2.06; −0.32] (SD: 0.74), mean width difference −0.45 mm [−0.76; −0.25] (SD: 0.19) and mean height difference was 0.41 mm [0.14; 0.62] (SD: 0.18). Although the height was increased in all models, both the length and width were systematically decreased, resulting in an average decrease of volume of −7.1 cm³ [−7.45; −6.77] (SD: 0.32). The number of triangles used to create each mesh ranged from 20944 to 368244, resulting in a variation of the file size from 1023 Ko to 80462 Ko (0.16 to 12.70 times the file size of the reference model). Two of the free programs created meshes with errors, such as the presence of holes (non-watertight meshes) that could be repaired with Netfabb.

Free programs able to convert volume imaging data to a printable virtual mesh do not provide equivalent results. Variations were noted in the three plane of space with a systematic difference between free programs and the commercial FDA-approved one. While the length and width were less than a millimeter different to the reference, the dimension that most varied was the length with a difference reaching −2.06 mm with itk-Snap. Geometric data also varied significantly, the number of triangles composing the meshes being much different than the reference, resulting in variable file sizes. This traduces the fact that algorithms used by the programs are not the same. In the era of 3D printing made directly accessible in surgical departments, great attention should be paid to the accuracy of the models created with free software.

Used routinely in maxillofacial reconstructive surgery, the chondrocostal graft is also applied to hand surgery in traumatic or pathologic indications. The purpose of this overview was to analyze at long-term follow-up the radiological and histological evolution of this autograft, in hand and wrist surgery. We extrapolated this autograft technique to the elbow by using perichondrium.

Since 1992, 148 patients have undergone chondrocostal autograft: 116 osteoarthritis of the thumb carpometacarpal joint, 18 radioscaphoid arthritis, 6 articular malunions of the distal radius, 4 kienbock's disease, and 4 traumatic loss of cartilage of the proximal interphalangeal (PIP) joint. Perichondrium autografts were used in 3 patients with elbow osteoarthritis.

Magnetic Resonance Imaging (MRI) was performed in 19 patients with a mean follow-up of 68 months (4–159). Histological studies were performed on: i) perioperative chondrocostal grafts (n=3), ii) chondrocostal grafts explanted between 2 and 48 months after surgery (n=10), and iii) perioperative perichondrium grafts (n=2).

Whatever the indication, the reconstruction by a chondrocostal/ostochondrocostal or perichondrium graft yielded satisfactory clinical results at long-term follow-up. The main question was the viability of the graft.

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For rib cartilage grafting: The radiological study indicated the non-wear of the graft and a certain degree of ossification. The MRI and histology confirmed a very small degree of osseous metaplasia and graft viability. The biopsies showed neo-vascularization of the cartilage that had undergone morphological, constitutional and architectural changes. Comparison of these structural modifications with perioperative chondrocostal graft histology is in progress.

Despite the strong mechanical strain in the hand and wrist, chondrocostal graft is a biological arthroplasty that is trustworthy and secure over the long term, although it can cause infrequent complications inherent to this type of surgery. Despite the inevitable histological modification, the cartilage remains alive and is of satisfactory quality at long term follow-up and fulfills the requirements for interposition and reconstruction of an articular surface.

The perichondrium graft constitutes a new arsenal to cure cartilage resurfacing. The importance of perichondrium for the survival of the grafted cartilage, as previously reported, as well as its role in resurfacing, is being investigated.

Reconstructing mandibular and maxillary bone defects with free vascularized bone flaps requires to take into account the aesthetic and functional requirements to consider subsequent placement of dental implants. It implies a three-dimensional conformation of the bone fragment. This is usually done by making osteotomies on the bone harvested. The aim of our study was to evaluate the interest of virtual planning and 3D printing using free software and a consumer printer in this indication.

Invesalius® software (Technology of Information Renato Archer Center, Campinas, Brazil) was used to build virtual models from the patients' CT scan imaging data. The surgical procedure was planned using Meshmixer® (Autodesk, San Rafael, United States). Meshlab® software (Visual Computing Lab, Pisa, Italy) was used to design cutting guides for the flap harvest and modelling. 3D printing of these guides with a consumer printer (Ultimaker 2® Ultimaker B.V., Geldermalsen, the Netherlands) allowed the transfer of the planning to the operating room.

Three patients requiring mandibular reconstruction underwent an iliac crest free flap, a fibula free flap and a scapula free flap, and could benefit from this technique. In each case, the bone resection was performed virtually and the positioning of the bone available at the donor site was simulated on screen. This allowed to anticipate the position and orientation of the cutting planes on the bone flap. From the anatomy of the donor site and the cutting planes, harvest templates and cutting guides could be designed by computer. Planning the conformation of the bone flap to the recipient site has allowed an anatomical, aesthetic and functional reconstruction of the bone defect.

Surgeon-made virtual planning and “low cost” 3D printing helps harvest the bone flap and position and orient the osteotomies to adapt it to the defect. They provide, both the patient and the surgeon, reduced operative time and better anticipation of the result, particularly in the context of the maxillofacial reconstruction. Compared to commercially available custom-made devices, this technique allows the manufacture of the guides without delay and at a cheap price.

Amniotic membrane (AM) and amnion/chorion foetal membranes (ACM) are mainly composed of collagen & laminin layers and constitute relatively new materials to the dental market. They have proven effective for periodontal treatments such as Guided Tissue Regeneration (GTR) [1–3].

Based on our expertise in the field of lyophilisation & securisation of human bone allograft (Phoenix® process), we aimed to develop our own process applied to ACM and to control its in vivoefficacy in GTR indication.

Human placentas were donated under informed consent. ACM were separated from placenta and processed with a proprietary AMTRIX (TBF) Process. Resulting product was called ACMTRIX.

The effectiveness of ACMTRIX in GTR was evaluated using an in vivorat calvaria defect model as followed:

Empty defect (2 animals),

One animal per study group was sacrificed after 8 weeks, all others after 8 weeks.

Evaluations were performed by: macroscopic observations, X Ray micro-CT, and histological analysis.

For all groups using ACMTRIX, no major sign of inflammation were observed macroscopically and histologically. Moreover, bone tissue was already mature from 8 weeks and bone filling was slight to moderate.

The higher mean rate of mineralization was obtained for the group associating DBM cancellous block + ACMTRIX.

Although a xenogenic material, ACMTRIX was very well integrated without significant inflammatory reaction compared to empty defect and fully integrated in subcutaneous area.

The mineralization was superior with DBM cancellous block probably thanks to the stabilization of the material in the defect. Used alone, ACMTRIX has no osteogenic potential.

In conclusion, ACMTRIX has the potential to function as barrier for GTR and the unique properties associated with this material can augment its potential as a matrix for periodontal regeneration.

The management of maxillofacial injuries requires restoring the contours of the facial skeleton to achieve an aesthetic outcome. When fractures are simple, open reduction and rigid fixation with stock titanium osteosynthesis plates is usually sufficient. However, when the damage is more substantial (when the fracture is comminuted or in case of a bone defect) anatomical landmarks are lost and the reconstruction requires the use of titanium meshes. These meshes are usually modelled intraoperatively to restore the contours of the bone. This can be a tough and time consuming task in case of minimal invasive approach and intraoperative edema. When the injury is unilateral, printing a 3D anatomical model of the mirrored unaffected side is an easy way to accurately pre-bend the mesh preoperatively. With the emergence of “low cost” consumer 3D printers, the aim of our study was to evaluate the cost of this technique in a department of maxillofacial surgery.

The first part of the study was to evaluate free software solutions available online to determine which of these could be used to create 3D virtual models from the patients' volume imaging data, mirror the model and export an STL file suitable for 3D-printing with a consumer 3D-printer. The second part was to identify the desktop 3D-printers commercially available according to the different technology used, their prices and that of consumables required.

Five free software solutions were identified to create STL meshes of the patient's anatomy from thin slice CT scan DICOM data. Two more were available to repair, segment and mirror them to provide a clean STL file suitable for 3D printing with a desktop 3D printer. The prices of 2 different printers were then listed for each of the 3 additive manufacturing technologies available to date. Prices ranged from 2,299 € for the Ultimaker 2+© (Fuse Deposition Modeling, FDM), to 4,999 € for the Sintratec© printer (Selective Laser Sintering, SLS), the Formlabs 2© (stereolithography) being at an intermediate price of 3,299 €. Finally, the cost of the manufacture of a model was calculated for each of these printers. Considering a model of a supraorbital ridge printed to restore the anterior wall of the frontal sinus, the volume of the mesh is around 20 cm³. This represents a cost of less than 1 € with the FDM technology, 4.70 € with stereolithography and 1.50 € with the SLS printer.

Since patents of additive manufacturing have become part of the public domain, the cost of 3D printing technology has fallen drastically. Desktop printers are now an investment accessible to a surgery department and the cost of the material is low. This allows the surgeons, by the mean of free software, to directly create 3D models of their patients' anatomy, mirror them if needed and manufacture a template to pre-bend titanium meshes that will be subsequently sterilized for the surgery. Having the printer in the department reduces manufacturing lead times and makes this technique possible even for urgent cases.

Introduction:

20 cases of bone defect have been treated by the induced membrane technique avoiding allograft, microsurgery and amputation

Introduction

20 cases of bone defect have been treated by the induced membrane technique avoiding allograft, microsurgery and amputation

Summary

Properties of human amniotic membrane are particularly interesting. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating its association with a potentially osteoinductive scaffold.

Summary

Human amniotic membrane has interesting properties for regenerative medicine. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating: the necessity of its osteodifferentiation and the impact on immunogenicity; its optimal condition for storage.

Introduction: Humerus non union is unfrequent, and reported series short. New fixation with or without autograft remain the gold standard to achieve bone union in 95% of cases. But no report are published in case of failure of that new procedure. 9 patients with a failure of autograft in humerus non union have been treated by new fixation an adjonction of BMP

Matériel et méthodes: 9 patients with an average age of 53,8 yo (24–71) have been treated and followed prospectively for a minimum time of 3 years. The delay between the fracture and the secon procedure was 31 months (6–103). The number of procedure after the fracture fixation was 1,4 (1–5). In 6/9 cases a technical pitfall during the initial procedure was pointed. In 3/9 cases a radial palsy associated with the initial fracture, a septic condition of the non union, general risk factors of non union (diabetes, tabac) and a non collaborative patient were reported.

Bone union was defined as the continuity of 4/4 cortex on Xray (AP and sagital plane) and or with ct scan. Osigraft^® (BMP7) was implanted in the resected zone of non union which was fixed with 2 plates after reaming and decortication.

Résults: No complication have been reported. One case failed (septic non union, 3 procedures, very active patient). The 8 last patients achieved bone union with a delay of 11,1 mois (6–14) without any additive procedure. The 3 septic cases have been solved. Shoulder and elbow function were good without nerves complications.

Discussion:: Autograft remains the gold standard in term of treatment of non union. But nothing is reported in humerus non union if iliac crest autograft have failed to achieve non union. In such an indication (failure after an autograft) and in such a level (humerus can be shorten) a stable fixation an a growth factor allowed to solve resistant cases of non union even in septic conditions.

The failure of the initial treatment of the fracture (unstable fixation, unfilled bone’s defect) remain the main cause of non union.

Purpose: The bone marrow (BM) mesenchymatous stem cell (MSC) is an osteochondrogenic stem cell which produces fibroblastic colony forming units (fCFU) in vitro. The regenerative activity of MSC could be useful for repairing extensive bone loss. A specific method for isolating human MSC (hMSC) is based on the expression of a membrane molecule, the very late antigen 1 (VLA1), was developed in the laboratory for possible cell therapy. Sorted VLA1-positive cells generate 100% of the fCFU and thus contain all of the MSC. The method used to isolate MSC was adapted to the mouse to elaborate an in vivo model.

Material and methods: VLA1-positive cells were isolated by direct sorting of BALB/cByJ@Ico and C57BL/6J@Ico male mouse BM. Total BM cells and VLA1-positive and negative cells were submitted to in vitro characterisation tests: i) expression of stem-cell-specific markers determined by flow cytometry and ii) cloning efficacy (CE=number of fCFU per 105 seeded cells) after ten days culture. VLA-positive cells not tested in vitro were cryopreserved for later use in vivo. When thawed, the VLA1-positive cells from BALB/c mice were characterised in vitro as described above and injected intravenously into female BALB/c mice. After three months, the recipient mice were sacrificed and fluorescent in situ hybridisation was used to search for the injected cells (with Y chromosome) or their progeny in different tissues.

Results: Sorted VLA1-positive cells expressed stem-cell-specific markers (CD34, Scal). Culture of the VLA1- positive and negative fractions revealed that all the fCFU were found in the VLA1-positive fraction. In the BALB/c stem line, sorting enabled a 10-fold enrichment of fCFU compared with total BM cells. As the C57 stem line exhibited only 2-fold enrichment, the BALC/c line was used for the in vivo study. Moreover, cryopreservation did not alter the CE of VLA1-positive cells nor their expression of specific markers. We report the in vivo results.

Conclusion: The method used for specific sorting of hMSC can be applied to murine BM. The mouse thus appears to be a good model for preclinical research on bone reconstruction.