Development of bioinks for 3D printing microporous, sintered calcium phosphate scaffolds

AUTHORS

Sergio A. Montelongo, Gennifer Chiou, Joo L. Ong, Rena Bizios & Teja Guda

ABSTRACT

Beta-tricalcium phosphate (β-TCP)-based bioinks were developed to support direct-ink 3D printing-based manufacturing of macroporous scaffolds. Binding of the gelatin:β-TCP ink compositions was optimized by adding carboxymethylcellulose (CMC) to maximize the β-TCP content while maintaining printability. Post-sintering, the gelatin:β-TCP:CMC inks resulted in uniform grain size, uniform shrinkage of the printed structure, and included microporosity within the ceramic. The mechanical properties of the inks improved with increasing β-TCP content. The gelatin:β-TCP:CMC ink (25:75 gelatin:β-TCP and 3% CMC) optimized for mechanical strength was used to 3D print several architectures of macroporous scaffolds by varying the print nozzle tip diameter and pore spacing during the 3D printing process (compressive strength of 13.1 ± 2.51 MPa and elastic modulus of 696 ± 108 MPa was achieved). The sintered, macroporous β-TCP scaffolds demonstrated both high porosity and pore size but retained mechanical strength and stiffness compared to macroporous, calcium phosphate ceramic scaffolds manufactured using alternative methods. The high interconnected porosity (45–60%) and fluid conductance (between 1.04 ×10−9 and 2.27 × 10−9 m4s/kg) of the β-TCP scaffolds tested, and the ability to finely tune the architecture using 3D printing, resulted in the development of novel bioink formulations and made available a versatile manufacturing process with broad applicability in producing substrates suitable for biomedical applications.

Flexible Osteogenic Glue as an All-In-One Solution to Assist Fracture Fixation and Healing

AUTHORS

Jincheng Tang, Kun Xi, Hao Chen, Lingjun Wang, Dongya Li, Yun Xu, Tianwen Xin, Liang Wu, Yidi Zhou, Jiang Bian, Zhengwei Cai, Huilin Yang, Lianfu Deng, Yong Gu, Wenguo Cui, Liang Chen

ABSTRACT

Osteogenic glue that reproduces the natural bone composition represents the final frontier of orthopedic adhesives with the potential to revolutionize surgical strategies against comminuted fractures. However, it is difficult to achieve an all-in-one formula, which could provide flexible and reliable adhesiveness while avoiding interfering with or even promoting the healing of glued fractures. Herein, an osteogenic glue characterized by inorganic-in-organic integration between amine-modified mesoporous bioactive glass nanoparticles (AMBGN) and bioadhesive gelatin-dextran network (GelDex) is introduced as an all-in-one tool to flexibly adhere and splice bone fragments and subsequently guide fracture healing during degradation. Relying on such integration, a 4-fold improvement in cohesiveness is presented, followed by a nearly 5-fold enhancement in adhesive strength in ex vivo porcine bone samples. The reversible and re-adjustable adhesiveness also enables glue to effectively splice intricate fragments from highly comminuted fractures in the rabbit radius in an in vivo environment. Moreover, well-preserved organic–inorganic integrity during degradation of the glue guides sustained interfacial osteogenesis and achieve satisfying healing outcomes in glued fractures, as observed by the 2-fold improvement in biomechanical and radiological performance compared with commercially available cyanoacrylate adhesives. The current findings propose an all-in-one solution for the fixation of bone fragments during surgery.

Osteolineage depletion of mitofusin2 enhances cortical bone formation in female mice

AUTHORS

Allahdad Zarei, Anna Ballard, Linda Cox, Peter Bayguinov, Taylor Harris, Jennifer L. Davis, Philip Roper, James Fitzpatrick, Roberta Faccio, Deborah J. Veis

ABSTRACT

Mitochondria are essential organelles that form highly complex, interconnected dynamic networks inside cells. The GTPase mitofusin 2 (MFN2) is a highly conserved outer mitochondrial membrane protein involved in the regulation of mitochondrial morphology, which can affect various metabolic and signaling functions. The role of mitochondria in bone formation remains unclear. Since MFN2 levels increase during osteoblast (OB) differentiation, we investigated the role of MFN2 in the osteolineage by crossing mice bearing floxed Mfn2 alleles with those bearing Prx-cre to generate cohorts of conditional knock out (cKO) animals. By ex vivo microCT, cKO female mice, but not males, display an increase in cortical thickness at 8, 18, and 30 weeks, compared to wild-type (WT) littermate controls. However, the cortical anabolic response to mechanical loading was not different between genotypes. To address how Mfn2 deficiency affects OB differentiation, bone marrow-derived mesenchymal stromal cells (MSCs) from both wild-type and cKO mice were cultured in osteogenic media with different levels of β-glycerophosphate. cKO MSCs show increased mineralization and expression of multiple markers of OB differentiation only at the lower dose. Interestingly, despite showing the expected mitochondrial rounding and fragmentation due to loss of MFN2, cKO MSCs have an increase in oxygen consumption during the first 7 days of OB differentiation. Thus, in the early phases of osteogenesis, MFN2 restrains oxygen consumption thereby limiting differentiation and cortical bone accrual during homeostasis in vivo.

Deletion of Ulk1 inhibits neointima formation by enhancing KAT2A/GCN5-mediated acetylation of TUBA/α-tubulin in vivo

AUTHORS

Changhan Ouyang, Jian Li, Xiaoxu Zheng, Jing Mu, Gloria Torres, Qilong Wang, Ming-Hui Zou & Zhonglin Xie

ABSTRACT

ULK1 (unc-51 like autophagy activating kinase) has a central role in initiating macroautophagy/autophagy, a process that contributes to atherosclerosis and neointima hyperplasia, or excessive tissue growth that leads to vessel dysfunction. However, the role of ULK1 in neointima formation remains unclear. We aimed to determine how Ulk1 deletion affected neointima formation and to investigate the underlying mechanisms. We measured autophagy activity, vascular smooth muscle cell (VSMC) migration and neointima hyperplasia in cultured VSMCs and ligation-injured mouse carotid arteries from male wild-type (WT, C57BL/6 J) and VSMC-specific ulk1 knockout (ulk1 KO) mice. Carotid artery ligation in WT mice increased ULK1 protein expression, and concurrently increased autophagic flux and neointima formation. Treating human aortic smooth muscle cells (HASMCs) with PDGF (platelet derived growth factor) increased ULK1 expression, activated autophagy, and promoted cell migration. Further, smooth muscle cell-specific deletion of Ulk1 suppressed autophagy, inhibited VSMC migration, and impeded neointima hyperplasia. Mechanistically, Ulk1 deletion inhibited autophagic degradation of histone acetyltransferase protein KAT2A/GCN5 (K[lysine] acetyltransferase 2A), resulting in accumulation of KAT2A that directly acetylated TUBA/α-tubulin and subsequently increased protein levels of acetylated TUBA. The acetylation of TUBA increased microtubule stability and inhibited VSMC directional migration and neointima formation. Finally, local transfection of Kat2a siRNA decreased TUBA acetylation and prevented the attenuation of vascular injury-induced neointima formation in ulk1 KO mice. These findings suggest that Ulk1 deletion inhibits neointima formation by reducing autophagic degradation of KAT2A and increasing TUBA acetylation in VSMCs.

The quality of etched enamel in different regions and tooth types and its significance in bonding and the development of white spot lesions

AUTHORS

Elisabeth C. Barnhart; Phillip M. Campbell; Amal Noureldin; Katie Julien; Peter H. Buschang

ABSTRACT

Objectives

To quantify differences in the etch quality of enamel within and between human teeth, which has not previously been attempted.

Materials and Methods

The buccal right and left halves of 27 extracted human teeth were randomly allocated to scanning electron microscopy (SEM) or micro–computed tomography (μCT) for evaluation. The buccal surfaces were pumiced, etched with 37% phosphoric acid gel etchant for 15 seconds, rinsed, and air dried. Each tooth was divided into three regions (incisal, middle, and cervical) and viewed after etching at 1200× magnification with SEM. The μCT scans were taken before and after etching to calculate apparent and material mineral densities.

Results

SEM showed greater aprismatic enamel and poorer etch quality (ie, significantly less percentage enamel) for the posterior than anterior teeth and for the cervical region than for the incisal and middle regions of all teeth. Although there were no density differences prior to etching, μCT demonstrated that etching increased material density significantly more for the anterior than posterior teeth. Prior to etching, the enamel in the cervical regions was significantly less dense than the enamel in the middle or incisal regions. Etching significantly increased the material density of all three regions, which decreased initial regional differences. After etching, the apparent density of the cervical region remained significantly lower than the densities of the other two regions.

Conclusions

Based on SEM and μCT, there is greater aprismatic enamel and inferior etch quality in the cervical regions of all tooth types and is clinically significant in explaining the failure of sealant retention and the propensity for white spot lesions.

RhoA/Rock activation represents a new mechanism for inactivating Wnt/β-catenin signaling in the aging-associated bone loss

AUTHORS

Wei Shi, Chengyun Xu, Ying Gong, Jirong Wang, Qianlei Ren, Ziyi Yan, Liu Mei, Chao Tang, Xing Ji, Xinhua Hu, Meiyu Qv, Musaddique Hussain, Ling-Hui Zeng & Ximei Wu

ABSTRACT

The Wnt/β-catenin signaling pathway appears to be particularly important for bone homeostasis, whereas nuclear accumulation of β-catenin requires the activation of Rac1, a member of the Rho small GTPase family. The aim of the present study was to investigate the role of RhoA/Rho kinase (Rock)-mediated Wnt/β-catenin signaling in the regulation of aging-associated bone loss. We find that Lrp5/6-dependent and Lrp5/6-independent RhoA/Rock activation by Wnt3a activates Jak1/2 to directly phosphorylate Gsk3β at Tyr216, resulting in Gsk3β activation and subsequent β-catenin destabilization. In line with these molecular events, RhoA loss- or gain-of-function in mouse embryonic limb bud ectoderms interacts genetically with Dkk1 gain-of-function to rescue the severe limb truncation phenotypes or to phenocopy the deletion of β-catenin, respectively. Likewise, RhoA loss-of-function in pre-osteoblasts robustly increases bone formation while gain-of-function decreases it. Importantly, high RhoA/Rock activity closely correlates with Jak and Gsk3β activities but inversely correlates with β-catenin signaling activity in bone marrow mesenchymal stromal cells from elderly male humans and mice, whereas systemic inhibition of Rock therefore activates the β-catenin signaling to antagonize aging-associated bone loss. Taken together, these results identify RhoA/Rock-dependent Gsk3β activation and subsequent β-catenin destabilization as a hitherto uncharacterized mechanism controlling limb outgrowth and bone homeostasis.