29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Prof. Dr.

Chair

Stability of biofunctionalized high-performance ceramics against 25 kGy gamma sterilization

Surface modification technologies

Implant associated

Abstract text (incl. figure legends and references) Introduction: Although high-performance ceramics (HPC) like alumina and zirconia exhibit excellent wear resistance, they provide poor osseointegration capacity. To improve this issue, new techniques have been successfully developed for HPC surfaces and stable cell adhesion can be achieved by covalently bound peptides. Objectives: We show that biofunctionalized surfaces can be sterilized without loss of functionality. Materials and Methods: For modification of alumina‑toughened zirconia, a 3-aminopropyldiisopropylethoxysilane monolayer was applied and coupled with cyclo-RGD (cRGD) peptides by using the crosslinker BS³. Samples were sterilized using 25 kGy gamma sterilization (GS) after each step of the biofunctionalization. Stability was investigated initially by contact angle measurement. To test functionality after sterilization, all missing functionalization steps were completed. Functionality was then demonstrated using human mesenchymal stem cells (MSC) in proliferation tests and cytotoxicity assays. Immunofluorescence staining (pFAK, Actin) was conducted to evaluate the adhesion of MSC on the surfaces. Results: It was shown that there is no significant difference between samples functionalized with cRGD before and after GS regarding contact angle, cytotoxicity, and pFAK activity. Sterilized samples showed significantly higher proliferation rates compared to non-sterilized controls. Samples sterilized after silanization or crosslinking showed no significant differences regarding their cytotoxicity and contact angle before or after GS. However, a significant decrease after GS was shown regarding cell proliferation and pFAK activity. Conclusion: The investigated functionalization enables improved adhesion, and proliferation of MSC. It was shown that fully sterilized samples are stable against GS. This is of importance since having a sterile product is a prerequisite to start translation steps towards preclinical and subsequently first-in-man applications.

Short Talk

Proteomic insights into personalised polycaprolactone (PCL) scaffold-guided diabetic bone healing

Tissue regeneration/regenerated medicine

Abstract text (incl. figure legends and references) Despite advancements in treating complex bone fractures, impaired healing remains a common complication in patients comorbid with type 2 diabetes mellitus (T2DM). The interdisciplinary research consortium "SyMBoD" aims to address this issue by studying the dynamics between T2DM and PCL scaffold-guided fracture healing through in silico modeling and in/ex vivo evaluation of diabetic bone healing, including histologic, proteomic, and metabolomic analyses of blood and tissue. Using a diabetic rat model with a critical-size femur defect, scaffold-supported regenerated and contralateral tissue at 21 or 42 days post-implantation was explanted and extracted for their metabolome and proteome. LC-MS/MS-based proteomic analyses were conducted on a quadrupole-Orbitrap mass spectrometer. Altered protein expression profiles were determined by pairwise comparisons of disease conditions and time points. Finally, results based on imaging information of longitudinal µCT and histological data were integrated. By employing a novel multi-omics extraction protocol combined with multiplexed mass tagging, over 4,000 proteins were reproducibly quantified in 4 animals per disease group and per time point, allowing for a comprehensive description of bone healing. Functional analysis of differentially expressed proteins confirmed that the systemic diabetic state also impairs the metabolism in bone, entailing a dysregulated healing. More precisely, diabetic animals showed prolonged expression of inflammatory proteins at both secretory and cellular levels, while expressing fewer structural proteins of a soft callus tissue. Matching the protein profiles to the imaging data further supported this observation. In summary, utilizing deep proteomic profiling of PCL-guided healing, this study unveils disturbed molecular processes of diabetic bone regeneration. It sets the groundwork for targeted scaffold optimization via biofunctionalization to address observed imbalances in healing pathways.

Comparative analysis of blood and synovia samples after implantation of new polymeric scaffolds in osteochondral defects of a sheep model

Abstract text (incl. figure legends and references) Treatment of osteochondral defects are still a challenge in the clinical situation of orthopedics and trauma surgery. Therefore, our BMBF funded consortium (FKZ 13XP5089) established new porous scaffolds of poly-((D,L)-lactide-ε-caprolactone)-dimethacrylat (LCM) by laser-assisted two-photon polymerisation that mimics the osteochondral region. Additionally, in one test group the pores were filled with biogel based on collagen for improved cellular migration. The present study aims to analyze the in vivo biocompatibility of biogel filled LCM compared to pure LCM scaffolds in an ovine model of osteochondral defect repair. LCM scaffolds with 7 mm diameter and 10 mm height were filled with biogel prior to implantation into osteochondral knee defects in the distal femur of adult female sheep (n=5). Before surgery and at day 5, 15, 30, 60, and 90 after implantation, sera were collected and analyzed by C-reactive protein (CRP) and lactate dehydrogenase (LDH) assay. Synovial fluid was harvested of the operated as well as of the untreated knee on day 90 and extensively analyzed. Before implantation, no significant difference were measured in CRP and LDH of sera in both groups. After insertion of LCM scaffolds, the CRP increased at the early time points and then decreased significantly to the late time points. Pure LCM scaffolds resulted in lower CRP concentrations in sera and synovial fluid whereas LCM+biogel scaffolds generated reduced LDH values. However, a higher LDH was found in synovia of operated compared untreated knee joint for both groups. Filling with biogel reduced the degradation of LCM and therefore the production of lactate. However, implantation of LCM+biogel resulted in upregulated CRP values that indicate an increased inflammatory reaction that might either be a sign of foreign body reaction or first stage of physiological defect healing. Thus, detailed analyses of the explanted scaffolds and surrounding tissue are necessary for further interpretation.

PD Dr.

Healing of critical size bone defects by mechano-hybrid-scaffolds with bioactive architecture

Additive manufacturing (e. g. 3D printing)

Abstract text (incl. figure legends and references) Until today, no pure biomaterial strategy for the regeneration of large bone defect exists despite clear advantages over the use of autologous bone grafts and growth factors. We have previously shown that a soft, collagen-based biomaterial with channel-like pores is able to induce development-like healing of critical size bone defects (Petersen et al., Nat Commun 2018). Here, we report on the incorporation of a 3D printed support structure for the mechanobiological optimization of the biomaterial and its evaluation in small and large animal models. Support structures were produced from medical grade polycaprolactone (PCL) by selective laser sintering, immersed into a collagen dispersion, directionally frozen and freeze-dried to produce mechano-hybrid-scaffolds (MHS). Mechano-bioreactors were used to characterize cell recruitment into MHS and to verify stability under in vivo-like loads. MHS were first evaluated in 5mm critical size defects in the rat femur and then applied to 3cm defects in the sheep tibia. No bioactive molecules, cells or bone grafts were added. Bone defect healing was studied via x-ray, µ-CT and (immune)histology. The strong cell recruitment potential of the collagen-based guiding structure was not impaired in MHS while mechanical properties were clearly improved. Implantation of MHS into the bone defect in rats revealed a robust induction of endochondral ossification. Compared to pure collagen scaffolds, MHS further improved the linear alignment of extracellular matrix fibers and stabilized the endochondral healing process. Nine weeks after implantation, three out of six animals were in the process of bony bridging. Even in the 3cm tibia defect in sheep, MHS induced a linear alignment of collagen fibers across the entire bone defect guiding tissue mineralization. The results of this study verify the potential of the architecturally and mechanobiologically optimized MHS to induce the regeneration of critical size bone defects.

Professor

Rational engineering of glycosaminoglycan-based dickkopf-1 scavengers to improve bone regeneration

Clinical applications and translation

Abstract text (incl. figure legends and references) Introduction: The WNT signaling pathway is a key regulator of bone development and regeneration comprising ligands, receptor and inhibitors. Functional alterations of WNT ligands and inhibitors are linked to bone diseases affecting bone fragility and resulting in a high medical and socioeconomic burden. Hence, this cellular pathway has emerged as a novel target for bone-protective therapies, e.g. in osteoporosis [1, 2]. Antibodies have been successful for neutralizing inhibitors but their application is limited due to potential cardiovascular risk [3]. Objectives: The aim was to investigate glycosaminoglycan (GAG) recognition by Dickkopf-1 (DKK1), a potent endogenous WNT inhibitor, and the underlying functional implications to develop novel WNT signaling regulators [4]. Materials and methods: In an interdisciplinary approach we combined in silico structure-based de novo design strategies and molecular dynamics simulations with synthetic chemistry and surface plasmon resonance spectroscopy to rationally engineer oligomeric GAG derivatives (REGAG) with improved neutralizing properties for DKK1. These molecules were validated in vitro in a Wnt-responsive cell line and in vivo in a mouse calvaria defect model with critical size bone lesions after absorbing to gelatin sponges [4]. Results: In vitro Alpl gene expression, Wnt reporter assay and in vivo analyses showed that the REGAGs modification translated into increased WNT pathway activity and improved bone regeneration. Importantly, REGAG outperformed polymeric high-sulfated hyaluronan, in enhancing bone healing due to improved DKK1 scavenging properties [4]. Conclusion: REGAG may represent an innovative strategy to develop novel therapeutic approaches for regenerative medicine [4]. [1] Saag KG et al. 2017, N Engl J Med., 377(15):1417-1427 [2] Florio M et al. 2016, Nat Commun. 7:11505. [3] Chames P et al. 2009, Br J Pharmacol. 157(2):220-33. [4] Ruiz-Gomez G et al. 2023, Biomaterials 297, 122105

Lecture hall 6

Short Talks 5

Bone Substitutes and Regeneration 1

Short Talk Session

Short Talks 5
Bone Substitutes and Regeneration 1

Appointment

Chair

Programme

Short Talks 5Bone Substitutes and Regeneration 1

Appointment

Chair

Programme

Short Talks 5
Bone Substitutes and Regeneration 1