In Vitro Coculture of Primary Human Cells to Analyze Angiogenesis, Osteogenesis, and the Inflammatory Response to Newly Developed Osteosynthesis Material for Pediatric Maxillofacial Traumatology: A Potential Pretesting Model before In Vivo Experiments

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Introduction
Te treatment of pediatric maxillofacial fractures remains a surgical challenge since fractures in children difer signifcantly from fractures in adults [1][2][3].Tose diferences include general skull and face anatomy, continuously growing and developing bones, bone structure of children, tooth development, and present tooth germs as well as difculties in cooperation of the patient during the treatment procedure [4].Te reconstruction of pediatric fractures requires in the frst instance the stabilization of the fragments but, in addition, must not impair the physiological growth of the bones.However, for open reduction and internal fxation (ORIF), nonresorbable titanium osteosynthesis plates still represent the gold standard on account of their excellent biocompatibility and stability [1,4,5].Nevertheless, the major disadvantage of the application of these plates is the need to be removed in a second operation very soon after the restoration, thus making a second surgical procedure necessary.Te latter carries the additional risk of injury to aesthetically and functionally important structures as well as the risk of scarring, especially in the facial area.As a result, the challenge in pediatric traumatological care is the requirement for resorbable and stable osteosynthesis materials that ensure the necessary stability over a defned period of time when treating fractures in the child's facial skeleton.In addition, the material must degrade in a time frame that avoids a second surgical operation procedure [6][7][8].Tus, to meet the clinical requirements, the material design should precisely correspond to the child's anatomical features by means of predetermined breaking points and fber reinforcement.In order to meet these requirements, during this study, a newly developed osteosynthesis material engineered from PDLLA (poly(D, L-lactide)) with difering combinations with calcium carbonate (CC), magnesium (Mg), and chitosan (CH) has been developed and evaluated in vitro.Te latter involved an endothelial/osteoblast coculture for bone tissue as the frst step for the in vitro determination of the optimal composition of these novel materials in the feld of pediatric traumatology.
Since a better understanding of bone fracture healing mechanisms can be generally assisted by in vitro studies, tissue-engineered human bone equivalents might signifcantly contribute to the understanding and improvement of developing new benefcial osteosynthesis materials in the context of pediatric maxillofacial fracture treatment [9][10][11].By means of innovative in vitro coculture models consisting of primary osteoblasts (or fbroblasts) and primary endothelial cells, a complex and broad evaluation of newly developed materials is possible depending on the material application [12][13][14].In the present basic research study, the coculture model for bone tissue consisted of human primary osteoblasts (pOB) and human primary dermal microvascular endothelial cells (HDMEC), which was then applied to fully characterize and evaluate the novel composite osteosynthesis material described previously.Besides general biocompatibility and cytotoxicity analyses, the more elaborate assessment of the materials' functionality with a focus on cellular processes associated with the implantation procedure, including infammation, vascularization, and osteogenic diferentiation, can be well achieved with this in vitro bone mimic model.
Various mechanisms are activated in the human organism after the implantation of a biomaterial [15][16][17].Te initial reaction that takes place when a biomaterial is implanted into the human body is a nonspecifc infammatory response that can range from good tolerability (biocompatibility) with a mild infammatory reaction through a rejection reaction to complete integration of the material in the optimal case.In order to be able to make a reliable statement about the body's infammatory reaction in response to the materials when coming into contact with the diferent materials, infammation-associated factors have been analyzed at both gene and protein levels in the in vitro model system described in the present study.Furthermore, the rapid supply of blood vessels (vascularization) is considered to be one of the key factors in bone regeneration and is therefore crucial for a good integration of the material.Numerous published studies have shown that the cocultivation of osteoblasts and endothelial cells reliably leads to the formation of microvessel-like structures with a functional lumen [18][19][20][21].To test the capability of vascularization of the material and to be able to make statements about the functionality of the material, the efect of the diferent materials on the endothelial ability to form microvessel-like structures as well as the expression and release of proangiogenic factors was investigated in the bone coculture model.In addition, with the use of this coculture model, the process of osteogenic diferentiation, which is also vital for sufcient fracture healing, can also be simulated in a meaningful way.To achieve this, the mineralization capacity as well as the relative gene expression of osteogenic diferentiation factors was assessed in the in vitro model in response to the diferent materials.Te following study using the coculture model as a biological mimic of bone tissue will demonstrate the model's efectiveness as a novel and innovative approach to evaluate new and highly sophisticated materials for pediatric traumatological care.

Ethical Statement.
All cells that were used for this study were obtained from excess tissue, and their application was in accordance with the principle of informed consent and approved by the responsible Ethics Commission of the state Hessen, Germany.

Material Composition.
For the frst evaluation step, the following basic materials were initially tested in terms of biocompatibility: PDLLA (poly(D, L-lactide)), PDLLA with calcium carbonate (PDLLA: CC), PDLLA with calcium carbonate and magnesium (PDLLA: CC + Mg), poly L-lactide and polyglycolic acid (PLLA: PGA) and chitosan (CH).Te base polymers were used as received: PDLLA and PLLA: PGA from Evonik (Darmstadt, Germany) and biocomposite material PDLLA: CC from SchäferKalk (Hahnstätten, Germany).Magnesium alloy WE43 was used as powder formulation (MeoTech, Aachen, Germany) and combined in a speed mixer (Hauschild, Hamm, Germany) at room temperature with mixing parameters (800 to 1200 rpm) in a fnal weight% formulation of 90% PDLLA: CC and 10% WE43.Chitosan raw material (DD � 90%) with a molecular weight of 110 kDa was manufactured and processed by the project-afliated partner BioLog Heppe ® GmbH [22].Terefore, chitosan was dissolved in acetic acid before starting the spinning process using a solvent wetspinning machine (FOURN É, Alfter, Germany).For initial biocompatibility analyses, fbers of chitosan were manufactured in multiflaments (Yarn count: 167 tex, diameter: For the osteosynthesis material combination with chitosan, the corresponding basis material that forms the matrix, PDLLA: CC, and the magnesium alloy was frst weighed and mixed in the speed mixer.Te chitosan endless fber was stretched in the press frame before the mixed composite material was placed on the chitosan continuous fbers.At a temperature of 80-120 °C and pressure of 100-200 bar for 10-30 minutes, the composite material was pressed and a wafer was produced.Te wafer was further machined in a milling device (Chiron, Tuttlingen, Germany) and was milled into coin shape structure for cell culture application in 24-well plates.
2.3.Primary Cells.Human primary osteoblasts (pOB) were isolated from juvenile cancellous bone fragments as already described [23].Cells were cultivated in Dulbecco's modifed Eagles' medium/nutrient mixture F-12 (Sigma-Aldrich, St. Louis, USA) supplemented with 10% FBS (Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, USA) at 37 °C in a humidifed atmosphere and were used up to passage 4 in this study.Human dermal microvascular endothelial cells (HDMECs) and human dermal fbroblasts (HDFs) were isolated from excess tissue of juvenile patients that underwent cleft lip reconstruction.Cells were isolated according to an established protocol [24] via several steps of enzymatic digestion and magnetic bead cell separation for endothelial cell-specifc CD31.Following cell separation, HDMECs were cultivated in an endothelial growth medium (Promocell) and were used up to passage 4. Human dermal fbroblasts were obtained as the CD31negative cell fraction and were further cultivated in Dulbecco's modifed Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA).All primary cell types were characterized in a standard protocol using cell-specifc immunofuorescence staining before they were used for cell experimentation.Cell culture experiments were performed with at least 3 diferent donors of primary cells (n).Te individual number of samples and donors is documented in the appropriate fgure legends of the results part.

Biocompatibility Analyses.
To exclude possible general cytotoxic or cell-damaging efects arising from the diferent basic material compositions (PLLA: PGA, PDLLA, PDLLA: CC, PDLLA: CC + Mg, and Chitosan) on the described cell types, an extraction assay on monocultures of the relevant primary cells (HDF, pOB, HDMEC) was performed prior to coculture experimentation (Figure 1(a), graphical representation).Tus, the diferent materials were incubated in a cell culture medium for 4 days at 37 °C in an atmosphere of 5% CO 2 and 95% oxygen.Primary fbroblasts, primary osteoblasts, and HDMEC were seeded on 96-well plates (5.000 cells/well).200 µl of the leached medium (extraction medium) was added to the preseeded cells, and cells were incubated for an additional four days before analyzing for cell viability using MTS CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay (Promega, Madison, USA) according to the manufacturer's protocol.Absorbance was measured at 490 nm in a microplate reader (GENios plus, TECAN, Crailsheim, Germany).Furthermore, an LDH assay was performed according to the manufacturer's protocol (Sigma-Aldrich, St. Louis, MO, USA) to determine the amount of dead cells after incubation of the cells with the leached medium of the diferent basic materials.

Adhesion Assay.
For initial material evaluation, the adhesion capacity of the appropriate cells in monoculture (HDMECs, pOB, and HDF) on the diferent basic materials was analyzed (Figure 1(a), graphical representation).In achieving this, PLLA: PGA, PDLLA, PDLLA: CC, and PDLLA: CC + Mg as well as solely chitosan fbers were placed in 24-well plates before the primary cells were seeded on top of the materials.Cell/material complexes were cultivated for 7 days before cells were fxed using 3.7% PFA and further processed for cell-type-specifc immunofuorescence staining and microscopic evaluation.

Coculture Experimentation.
For the in vitro coculture model for bone tissue, HDMEC and pOB were mixed in a 1 : 2 ratio and then seeded on top of the materials.
Coculture experimentation was performed to evaluate the materials' functionality of the newly manufactured materials combined with chitosan (CH): PLLA: PGA CH, PDLLA CH, PDLLA: CC + Mg CH, and PDLLA: CC CH (Figure 2(a), graphical representation).Te diferent materials were placed in 24-well plates before 20.000 pOB and 20.000 HDMEC were mixed and added to the materials without an additional coating of the materials and further cocultivated for 14 days.Due to the higher growth sensitivity of endothelial cells in the coculture system, cells were cultivated in an endothelial cell growth medium with medium change twice per week.After 14 days of cultivation, supernatants of coculture/material combinations were collected for further experimentation (ELISA) and stored at −80 °C until analyzation.In addition, cells were lysed and processed for mRNA isolation and quantitative real-time PCR as well as fxed for immunofuorescence staining (CD31) and alizarin red staining.During the course of cultivation, pH values were measured and documented in the supernatants of the diferent material-cell complexes using a pH measurement device (FiveEasy ™ FE20, Mettler-Toledo AG, Schwerzenbach, Switzerland) on day 1, day 7, and day 14 of cultivation.

Osteogenesis Assay.
To quantify the mineralization capacity of the cocultures seeded with the diferent materials, an osteogenesis quantifcation kit was used according to the manufacturer's protocol (Merck Millipore, Darmstadt, Germany).Terefore, after removing the material, cocultures at the bottom of the cell culture plastic were incubated with an alizarin red staining solution for at least 20 minutes before the excess dye was removed.Washed and stained cells were then assessed and documented using a light microscope.After visual inspection and documentation of the stained cells, alizarin red was removed from the cells via acetic acid (10%) before the stain was quantifed using a microplate reader (OD 405 ).Alizarin concentration was defned as µM.
2.9.Growth Factor and Cytokine Determination.Cell culture supernatants of the coculture/material complexes were collected after 14 days of cultivation and subsequently analyzed for growth factor and cytokine concentration using ELISA Development Systems according to the manufacturer's protocol (R&D Systems).For visualization of the protein content in the supernatants, a streptavidin-HRP colorimetric reaction was used, and the optical density was measured using a microplate reader (Tecan, Crailsheim, Germany) at a wavelength of 450 nm.Te following ELISA DuoSets were used in this study: vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), intercellular adhesion molecule 1 (ICAM-1), osteoprotegerin (OPG), and interleukin 8 (IL-8).

Gene Expression Analysis.
RNA isolation was performed using RNeasy micro kit according to the manufacturer's instructions (Qiagen).1 µg of extracted RNA was used to transcribe into complementary DNA (cDNA) according to a standard protocol using Omniscript Reverse Transcription Kit (Qiagen).For quantitative real-time PCR, the following primers were used during this study: Eselectin, osteonectin, and alkaline phosphatase (ALP).For quantitative real-time PCR (qRT PCR), 4 ng cDNA was used for one reaction and with the following cycler program: 95 °C 10 min, 95 °C 15 sec, 60 °C 1 min, 40 m cycles.To specify the length of the DNA fragments, a dissociation stage was added to the programme.qRT PCR was performed in triplicate, and relative gene expression was determined using the deltadelta CT method.Gene expression was compared by setting control cultures to 1 (reference value) as indicated in the relevant fgures.

Statistical Analyses.
All experiments were performed with at least three diferent donors of primary cells (n) as indicated in the appropriate fgure legends, and individual data points for each "n" are depicted in the diagrams.Results were calculated as mean ± standard deviation (SD) and were evaluated for signifcant diferences with one-way ANOVA and post-hoc testing (Dunnett's multiple comparison test) using GraphPad Prism 9.0 software (GraphPad Software Inc.).Statistically signifcant diferences (p values) were directly documented in the diagrams of the fgures.

Evaluation of Material Efects on Cell Viability and Cell-to-Material Adhesion of Primary Cells in
Monoculture: HDF, HDMEC, and pOB.To exclude possible cytotoxic efects arising from the diferently composed basic materials, the relevant primary cell types such as human dermal fbroblasts (HDFs), human primary osteoblasts (pOBs), and human dermal microvascular endothelial cells (HDMECs) were frst used for analyzation in monocultures, respectively.Terefore, the sterilized samples of the following basic materials (combination) PDLLA, PLLA: PGA, PDLLA: CC + Mg, PDLLA: CC, and chitosan were initially examined with respect to possible cytotoxic efects in monocultures of the appropriate cell types (Figure 1(a)).For this purpose, HDMEC, pOB, and HDF were cultivated separately in an extract of the diferent materials before the viability of the cells was determined using MTS assay and LDH assay (Figures 1(b) and 1(c)).No negative efects of the extraction media (leached medium) on the cells in monoculture could be determined when cells were cultivated in the diferent extraction media for 4 days.Te direct contact of the cells with the materials followed by cell-type-specifc immunofuorescence staining for the appropriate cell types (CD31, osteopontin, SMA) also confrmed consistently good cell-tomaterial adhesion and a regular cell-type-specifc morphology of all cell types seeded on the materials (Figure 1(d)).Physiological pH values ranging from pH 6.8 to pH 7.1 could be determined when cocultures were cultivated for 1, 7, and 14 days on all tested materials except for the PDLLA group when combined with the Mg alloy (Figure 2 materials displayed good biocompatibility and met the of an osteosynthesis material.Te lack of cytotoxicity permitted further analyses with regard to the functionality of the materials.

Evaluation of Infammation-Associated Factors in the In Vitro Coculture Model for Bone Tissue Seeded on Diferently
Composed Materials in Combination with Chitosan.For evaluation of material functionality, the diferent polymers PLLA: PGA, PDLLA, PDLLA: CC, and PDLLA: CC + Mg in combination with chitosan were characterized using a primary cell coculture model for bone tissue in vitro.For the functional evaluation, the coculture system consisting of primary osteoblasts (pOBs) and primary endothelial cells (HDMECs) was used.For the cell-biological characterization, the established coculture was incubated on the respective samples for 14 days before various tests were carried out with regard to three main functional parameters, namely, the processes of infammation, angiogenesis, and osteogenic diferentiation (graphical scheme in Figure 2(a)).
To investigate a possible material-mediated induction of an infammatory response of the coculture, the cell culture supernatants were analyzed for the proinfammatory factors ICAM-1 and IL-6 (Figures 2(d) and 2(e)).Although no signifcant diference could be observed in the appropriate cell culture supernatants, ICAM-1 was found to be less concentrated in PDLLA: CC + Mg CH and PDLLA: CC CH group supernatants compared to controls.A slight increase in ICAM-1 release could be assessed in supernatants of PLLA: PGA CH and PDLLA CH when combined with the coculture.Interleukin-6, another proinfammatory cytokine, was signifcantly lower in supernatants of coculture material complexes of PDLLA: CC + Mg CH (Figure 2(e)) compared to all other analyzed supernatants.In addition, relative gene expression of E-selectin was assessed in the appropriate cultivated cell-material complexes after 14 days of cultivation (Figure 2(f )) and revealed no signifcant diferences in relative quantifcation of the expression of E-selectin among the tested experimental groups.

Analysis of Microvessel-Like Structure Formation and Proangiogenic Growth Factor Production in the In Vitro Coculture Model for Bone Tissue Seeded on Diferently Composed Materials in Combination with Chitosan.
To evaluate the capability of the endothelial cells to form microvessel-like structures in the coculture system seeded on the diferent polymers combined with chitosan (PLLA: PGA CH, PDLLA CH, PDLLA: CC + Mg CH, PDLLA: CC CH), cocultures were cultivated for 14 days before cells were stained immunofuorescently for the endothelial marker CD31 to document the formation of microvessel-like structures in the coculture compared to control (Figures 3(a) and 3(b)).Although the immunostain of the coculture directly on the materials showed background exposure due to autofuorescence of the materials, angiogenic structures were clearly defned when cells were combined with PLLA: PGA CH and PDLLA CH as well as with PDLLA: CC CH (Figure 3(a), upper row).In contrast, no microvessel-like structure formation could be documented when cocultures were seeded on top of PDLLA: CC + Mg CH material (Figure 3(a), upper row).In this group, endothelial cells failed to form angiogenic structures and did not even exhibit the normal endothelial cell typespecifc morphology.In addition, the bottom of the cell culture plates of all experimental groups was also examined for possible CD31-positive cells that might have migrated from the material to the well plate.Te results confrmed those from the direct evaluation (Figure 3(a), lower row).Te induction of microvessel-like structure formation could be observed in cocultures of all analyzed experimental groups except for cocultures in combination with PDLLA: CC + Mg CH (Figure 3(a), lower row).Te total length of microvessel-like structures was comparatively quantifed using an image analysis program and confrmed the visual results from the immunostaining.Te combination of the coculture with PDLLA: CC CH revealed the highest value (total length in pixels) of microvessel-like structures compared to the other analyzed material-cell groups (Figure 3(c)).No formation of angiogenic structures could be quantifed in the PDLLA: CC + Mg CH group.Te determination of the proangiogenic factors VEGF and IL-8 in the cell culture supernatants of the diferent coculture/ material complexes revealed signifcant diferences among cocultures seeded on the diferent scafold and control cocultures (Figures 3(d) and 3(e)).Signifcantly higher release of VEGF into cell culture supernatants could be measured in the PDLLA: CC + Mg CH experimental group compared to all other groups, except for the VEGF release of the control pOB supernatants alone (Figure 3(d)).In contrast to the PDLLA: CC + Mg CH group, VEGF release of the cells seeded on the other tested materials ranged at a similar lower level.Analysis of the release of the proangiogenic cytokine interleukin-8 revealed the signifcantly lowest concentration in the PDLLA: CC + Mg CH group combined with the coculture and compared to all other analyzed experimental groups (Figure 3(e)).

Efect of Diferent Material Combinations with Chitosan on Osteogenic Diferentiation Capacity in the In Vitro Coculture Model for Bone Tissue: Mineralization and Osteogenic
Diferentiation Factor Analyses.Te determination of the mineralization of the osteoblasts as a marker for osteogenic diferentiation in the coculture dependent on the diferently composed materials PLLA: PGA CH, PDLLA: CH, PDLLA: CC + Mg CH was investigated using alizarin red staining of the cells grown on cell culture plastic with indirect contact to the material.A clear and signifcant initial mineralization of the osteoblasts in the coculture could be observed when cells were cultivated on PDLLA: CC CH and PDLLA: CC + Mg CH as documented by bright red areas showing the calcifed primary osteoblasts (arrows Figure 4(a)).No or less alizarin red-stained-positive calcifed cells could be detected in the control and when cells were combined with PLLA: PGA CH and PDLLA CH.Te visual results could also be confrmed by quantifcation of the alizarin stain (Figure 4(b)).Analysing the supernatants of the coculture/material comfor release of cocultivation revealed similar concentrations in all analyzed cell/material groups, except for the control endothelial cells in monoculture (Figure 4(c)).Relative quantifcation of gene expression profle documented a signifcant upregulation of the late osteogenic diferentiation marker osteonectin when cocultures were cultivated on PDLLA: CC CH and PDLLA: CC + Mg CH compared to the cultivation of the cells on the other materials and compared to the control (Figure 4(e)).Although the results were not signifcant, alkaline phosphatase (ALP) gene expression, an early marker for osteogenic diferentiation was found to be slightly upregulated in all analyzed coculture/material complexes compared to the control without material (Figure 4(d)).

Discussion
In the present basic research study, an in vitro coculture model for bone tissue was used to fully characterize and evaluate a newly developed osteosynthesis material engineered from PDLLA (poly(D,L-lactide)) together with calcium carbonate (CC), magnesium (Mg), and chitosan (CH) adapted to the specifc requirements of pediatric maxillofacial traumatology.However, to date, only a few osteosynthesis plates are available specifcally for use in pediatric care, and the development of osteosynthesis plates that are stable but can still be degraded quickly at defned points would be an important and forward-looking step in the pediatric treatment of facial fractures.Currently, there are few resorbable osteosynthesis plates available for clinical use.Tese material classes are made from polylactides (PLA), polyglycolides, and their copolymers [9,25].Due to their resorbability, these materials circumvent the disadvantages of titanium plates, especially the problem of growth inhibition and elimination of the need for another operation to remove the material.
Before a novel biomaterial can be used as a medical device, in vitro proof of its biocompatibility in mandatory cell culture tests according to defned international standard protocols is the frst essential step.However, currently proposed in vitro methods for this evaluation of biocompatibility fail to represent the physiological situation, since genetically modifed single-cell lines are generally used for the experiments and do not mirror the physiological situation in healthy tissue [26,27].During the present study, initial classical biocompatibility analyses that were performed with primary monocultures of primary osteoblasts, primary fbroblasts, and primary endothelial cells to make a frst selection were able to exclude cytotoxic efects of the pure basic materials composed of the polymer combined with calcium carbonate and/or magnesium alloy on the appropriate primary cells in monoculture.Cell-to-material adhesion of each cell type seeded separately on the basic materials confrmed the results of cytotoxicity assays by exhibiting overall good cell adhesion of the tested cell monocultures on all used materials.Although a single cell type can give a frst indication of the possible cytotoxicity of a tested material or one of its components, the prognostic statement on how the material will afect human tissue after implantation is very limited [28].Te integration of a biomaterial such as the here proposed osteosynthesis materials for application in pediatric maxillofacial traumatology strongly depends on the complex cellular interaction of diferent cell types that come into contact with the material after implantation.Currently, those cellular dynamics and cell interactions are still poorly represented when assessing a biomaterial in vitro.Nevertheless, to evaluate the material's functionality after implantation, in vitro tests require a cellular microenvironment that consists of more than one cell type and thus is more comparable to the physiological bone tissue.In this context, establishing and using an in vitro coculture model consisting of primary osteoblasts and primary endothelial cells can be strongly recommended to mimic the in vivo situation and might serve as a reliable bone replicate in vitro.Using this coculture model, the major fndings of the study were as follows: (1) none of the tested materials leads to a signifcantly increased release or upregulated gene expression of infammation-associated factors; (2) induction of vessel-like structures could be detected in all tested material/coculture groups except for the PDLLA: CC + Mg CH-coculture combination; and (3) signifcantly increased osteogenic diferentiation could be assessed solely in PDLLA: CC + Mg CH-cocultures as well as in PDLLA: CC CH-cocultures.
In general, a coculture is a very dynamic system, which is controlled by various growth factors and by the way cells communicate with each other similar to the in vivo situation, a process known as cellular crosstalk [21].During the last decade, several coculture systems related to bone regeneration have been developed [29][30][31][32].Especially, endothelial/osteoblast coculture models are able to simulate the physiological situation around the implant site, as both endothelial and osteoblastic cells are the key regulators for bone regeneration processes after trauma.In addition, endothelial/osteoblast coculture models represent a convenient way to study cell-biomaterial interactions with regard to the possible induction of infammatory processes.Te implantation of a biomaterial always involves tissue trauma which results in a physiological infammatory response, coupled with a wound healing and tissue regeneration reaction [33].Although any cytotoxic efect of the basic materials on the used cell types in monoculture could be excluded using biocompatibility and cellular adhesion assays, additional proinfammatory factors and cytokines were analyzed at gene and protein expression level in additional experiments to exclude possible prolonged tissue injury via chronic infammation after the implantation process.Te proinfammatory factors ICAM-1, interleukin-6 (Il-6), and E-selectin were consistently on a similar same level in all tested coculture/material groups or were even lower compared to control cocultures without material contact.Te release of proinfammatory factors such as endothelial-cellbased E-selectin is an excellent monitor of the body's early reaction to a biomaterial [34] and can thus be used to assess biomaterial components with respect to unwanted biological reactions.Tis gives valuable input for the development or improvement of the material.

10
Journal of Tissue Engineering and Regenerative Medicine Angiogenesis, the formation of new blood microvessels, in order to transport nutrients and oxygen to the implant site and is therefore initiated immediately after tissue injury [35].Microvascular endothelial cells are the basic component of blood vessels, and their function with regard to bone regeneration is essential for the formation of new vessels [36].When angiogenesis fails, peri-implant tissue will undergo ischemic changes.In the present study, the angiogenic capability of endothelial cells in the coculture consisting of primary HDMEC and primary osteoblasts revealed a clear induction of microvessel-like structure formation after 14 days of cocultivation.Tis was observed in the following tested material compositions: PDLLA CH, PDLLA: CC CH, and PLLA: PGA CH.In contrast, a lack of formation of microvessel-like structures was found when cocultures were cultivated on PDLLA: CC + Mg CH, only difering in the content of magnesium (Mg) that has been integrated within the material.Te included magnesium appears to lead to generally lower cell numbers of endothelial cells in the coculture, accompanied by a higher cell number of osteoblasts in this group compared to the other materials.Tis efect could be confrmed by a signifcantly higher release of pOB-derived VEGF in the coculture cultivated on PDLLA: CC + Mg CH scafolds as well as a signifcantly lower amount of endothelial cell type-specifc interleukin-8 release into the coculture supernatants.Optimal integration of an osteosynthesis material strongly depends on a good balance of cell proliferation and diferentiation of the diferent cell types in the peri-implant tissue.Terefore, the material composition must be chosen in such a way that neither of the two cell types has an advantage or disadvantage in cell growth, proliferation, and diferentiation.Te magnesium alloy that was included in the PDLLA: CC CH scafolds did not show any cytotoxic efect on the endothelial cells in monoculture but seemed to promote a signifcant selective advantage for the primary osteoblasts within the coculture systems.Tus, the cell ratio was skewed in favour of the osteoblasts, leading to a complete overgrowth by the pOB when cocultivated with HDMEC on PDLLA: CC + Mg CH scafolds.It is already known that magnesium-based biomaterials might increase the process of osteogenesis, promotion of osteoblast adhesion and motility, and positive immunomodulation, as well as angiogenesis [37].Te positive efect on osteogenic diferentiation of pOB in the coculture could be observed in the scafolds combined with the Mg alloy documented by positive alizarin red staining and quantifcation.Nevertheless, the highest induction of mineralization was found when cocultures were seeded on PDLLA: CC CH scafolds.Calcium carbonate is known to promote bone regeneration, as it can be converted into hydroxyapatite on coming into contact with phosphate solutions, thus serving as a source of apatite important for bone repair [38,39].Te osteoconductive qualities of calcium carbonate are well known, and it has been suggested that it can trigger stem cells or progenitors to diferentiate into osteoblasts [40,41].Although early osteogenic diferentiation markers were not signifcantly changed at protein and gene expression levels in the diferent coculture/material complexes, the relative gene expression of the late osteogenic marker, osteonectin, was found to be signifcantly upregulated in the calcium carbonate material groups, documenting an ongoing osteogenic differentiation process induced by those materials in the coculture.

Conclusion
By using the described coculture system consisting of primary osteoblasts and dermal microvascular endothelial cells, the authors were able to evaluate various material combinations of newly developed osteosynthesis materials for pediatric maxillofacial traumatology and could assess PDLLA: CC CH as the most functionally tested material.Te experimental study focused on biocompatibility, infammation, blood vessel formation, and osteogenic diferentiation.Complex in vitro models, like the proposed coculture bone mimic, allow a frst complete evaluation of the manufactured material, as the essential aspects occurring after implantation, especially infammation, angiogenesis, and osteogenic diferentiation, can be assessed in a more in vivo-like model than is possible with monocultures.Tis in turn deepens our understanding of biological and cellular reactions at the tissue-biomaterial interface and provides valuable input to help modulate and adapt the material to achieve the desired biological functions.Such models which permit cellular crosstalk are necessary for the further development and optimization of novel osteosynthesis materials for the special requirements in pediatric maxillofacial traumatology.

Figure 1 :
Figure 1: Evaluation of efects of basic material on cell viability and cell-to-material adhesion of primary cells in monoculture.(a) Graphical representation of experimental setting for initial evaluation of the materials PDLLA, PDLLA: PGA, PDLLA: CC + Mg, and Chitosan: biocompatibility analyses and cell adhesion assay were performed in monocultures of primary cells HDF, HDMEC, and pOB (n � 4).(b) Determination of relative cell viability compared to control using an MTS extraction assay (n � 4).(c) Analyzation of relative LDH amount in response to leached medium of the used materials compared to lysis control set to 100%.(d) Cell type-specifc immunofuorescence staining of pOB (Osteopontin) and HDF (SMA) an HDMEC (CD31) of cells seeded on top of the appropriate materials to assess cell-tomaterial adhesion (n � 4).Scale bar in fgure D � 150 µm.

Figure 2 :
Figure 2: Evaluation of infammation-associated factors in an in vitro coculture model for bone tissue seeded on diferently composed materials in combination with chitosan.(a) Schematic overview of experimental settings for in vitro coculture experimentation for functional evaluation (infammation, angiogenesis, and osteogenesis) of base material combined with chitosan (PDLLA: PGA CH, PDLLA CH, PDLLA: CC CH, and PDLLA: CC + Mg CH) (n � 4).(b) Overview of the material-coculture and monoculture combination in 24-well plates.(c) Determination of pH in cocultures seeded on the appropriate materials after 1, 7, and 14 days of cultivation (n � 3).(d) Evaluation of ICAM-1 in cell culture supernatants after 14 days of cultivation.(e) Evaluation of IL-6 in supernatants after 14 days of cultivation (n � 4).(f ) Relative quantifcation of gene expression of E-selectin in cocultures seeded on diferent materials after 14 days of cultivation (n � 3).
(c)).Although the material composition with PDLLA: CC + Mg CH (PDLLA: CC + Mg CH) led to the general increase in pH from ∼pH 7 to ∼pH 7.8 as demonstrated by discoloration of the pH indicator phenol red supplemented to the cell culture medium (Figure 2(b)) and fnally documented using a pH measurement device (Figure 2(c)) after 1, 7, and 14 days of cultivation, all analyzed

Figure 3 :Figure 4 :
Figure 3: Analysis of microvessel-like structure formation and proangiogenic growth factor production in an in vitro coculture model for bone tissue seeded on diferently composed materials in combination with chitosan.(a) Endothelial cell type-specifc immunofuorescence staining for CD31 of cocultures seeded on top of the material (upper row) and cells at the bottom of the cell culture plate (lower row) after 14 days of cocultivation (n � 4).(b) Control cocultures seeded on cell culture plastic without material (n � 4).(c) Quantifcation of the formation of microvessel-like structures (n � 4).(d) Evaluation of VEGF concentration in cell culture supernatants of cocultures seeded on the diferent materials compared to control cocultures as well as to the appropriate cells in monoculture (n � 4).(e) Evaluation of IL-8 concentration in cell culture supernatants of cocultures seeded on the diferent materials compared to control cocultures as well as to the appropriate cells in monoculture (n � 4).Scale bars � 150 µm.