Acoustic Patterning of Growth Factor for Three-Dimensional Tissue Engineering.

Temporal and spatial presentations of biological cues are critical for tissue engineering. There is a great need in improving the incorporation of bioagent(s) (specifically growth factor(s) [GF(s)]) onto three-dimensional scaffolds. In this study, we developed a process to combine additive manufacturing (AM) technology with acoustic droplet ejection (ADE) technology to control GF distribution. More specifically, we implemented ADE to control the distribution of recombinant human bone morphogenetic protein-2 (rhBMP-2) onto polycaprolactone (PCL)-based tissue engineering constructs (TECs). Three substrates were used in this study: (1) succinimide-terminated PCL (PCL-N-hydroxysuccinimide [NHS]) as model material, (2) alkali-treated PCL (PCL-NaOH) as first control material, and (3) fibrin-coated PCL (PCL-Fibrin) as second control material. It was shown that our process enables a pattern of BMP-2 spots of ∼250 µm in diameter with ∼700 µm center-to-center spacing. An initial concentration of BMP-2 higher than 300 µg/L was required to retain a detectable amount of GF on the substrate after a wash with phosphate-buffered solution. However, to obtain detectable osteogenic differentiation of C2C12 cells, the initial concentration of BMP-2 higher than 750 µg/L was needed. The cells on PCL-NHS samples showed spatial alkaline phosphatase staining correlating with local patterns of BMP-2, although the intensity was lower than the controls (PCL-NaOH and PCL-Fibrin). Our results have demonstrated that the developed AM-ADE process holds great promise in creating TECs with highly controlled GF patterning. Impact statement The combined process of additive manufacturing with acoustic droplet ejection to control growth factor (GF) distribution across three-dimensional (3D) porous scaffolds that is presented in this study enables creating 3D tissue engineering constructs with highly controlled GF patterning. Such constructs enable temporal and spatial presentations of biological cues for enhancing cell migration and differentiation and eventually the formation of targeted tissues in vitro and in vivo.

Functionally Graded, Bone- and Tendon-Like Polyurethane for Rotator Cuff Repair.

Critical considerations in engineering biomaterials for rotator cuff repair include bone-tendon-like mechanical properties to support physiological loading and biophysicochemical attributes that stabilize the repair site over the long-term. In this study, UV-crosslinkable polyurethane based on quadrol (Q), hexamethylene diisocyante (H), and methacrylic anhydride (M; QHM polymers), which are free of solvent, catalyst, and photoinitiator, is developed. Mechanical characterization studies demonstrate that QHM polymers possesses phototunable bone- and tendon-like tensile and compressive properties (12-74 MPa tensile strength, 0.6-2.7 GPa tensile modulus, 58-121 MPa compressive strength, and 1.5-3.0 GPa compressive modulus), including the capability to withstand 10 000 cycles of physiological tensile loading and reduce stress concentrations via stiffness gradients. Biophysicochemical studies demonstrate that QHM polymers have clinically favorable attributes vital to rotator cuff repair stability, including slow degradation profiles (5-30% mass loss after 8 weeks) with little-to-no cytotoxicity in vitro, exceptional suture retention ex vivo (2.79-3.56-fold less suture migration relative to a clinically available graft), and competent tensile properties (similar ultimate load but higher normalized tensile stiffness relative to a clinically available graft) as well as good biocompatibility for augmenting rat supraspinatus tendon repair in vivo. This work demonstrates functionally graded, bone-tendon-like biomaterials for interfacial tissue engineering.

Customized, degradable, functionally graded scaffold for potential treatment of early stage osteonecrosis of the femoral head.

Osteonecrosis of the femoral head (ONFH) is a debilitating disease that results in progressive collapse of the femoral head and subsequent degenerative arthritis. Few treatments provide both sufficient mechanical support and biological cues for regeneration of bone and vascularity when the femoral head is still round and therefore salvageable. We designed and 3D printed a functionally graded scaffold (FGS) made of polycaprolactone (PCL) and ß-tricalcium phosphate (ß-TCP) with spatially controlled porosity, degradation, and mechanical strength properties to reconstruct necrotic bone tissue in the femoral head. The FGS was designed to have low porosity segments (15% in proximal and distal segments) and a high porosity segment (60% in middle segment) according to the desired mechanical and osteoconductive properties at each specific site after implantation into the femoral head. The FGS was inserted into a bone tunnel drilled in rabbit femoral neck and head, and at 8 weeks after implantation, the tissue formation as well as scaffold degradation was analyzed. Micro-CT analysis demonstrated that the FGS-filled group had a significantly higher bone ingrowth ratio compared to the empty-tunnel group, and the difference was higher at the distal low porosity segments. The in vivo degradation rate of the scaffold was higher in the proximal and distal segments than in the middle segment. Histological analysis of both non-decalcified and calcified samples clearly indicated new bone ingrowth and bone marrow-containing bone formation across the FGS. A 3D printed PCL-ß-TCP FGS appears to be a promising customized resorbable load-bearing implant for treatment of early stage ONFH. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1002-1011, 2018.

Endothelial pattern formation in hybrid constructs of additive manufactured porous rigid scaffolds and cell-laden hydrogels for orthopedic applications.

Vascularization of tissue engineering constructs (TECs) in vitro is of critical importance for ensuring effective and satisfactory clinical outcomes upon implantation of TECs. Biomechanical properties of TECs have remarkable influence on the in vitro vascularization of TECs. This work utilized in vitro experiments and finite element analysis to investigate endothelial patterns in hybrid constructs of soft collagen gels and rigid macroporous poly(ε-caprolactone)-ß-tricalcium phosphate (PCL-ß-TCP) scaffold seeded/embedded with human umbilical vein endothelial cells (HUVECs) for bone tissue engineering applications. We first fabricated and characterized well-defined porous PCL-ß-TCP scaffolds with identical pore size (500µm) but different strut sizes (200 and 400µm) using additive manufacturing (AM) technology, and then assessed the HUVEC׳s proliferation and morphogenesis within collagen, PCL-ß-TCP scaffold, and the collagen-scaffold hybrid construct. Results showed that, in the hybrid construct, the cell population in the collagen component dropped by day 7 but then increased by day 14. Also, cells migrated onto the struts of the scaffold component, proliferated over time, and formed networks on the thinner struts (i.e., 200µm). Also, the thinner struts resulted in formation of long linear cellular cords structures within the pores. Finite element simulation demonstrated principal stress patterns similar to the observed cell-network pattern. It is probable that the scaffold component modulated patterns of principal stresses in the collagen component as biomechanical cues for reorganization of cell network patterns. Also, the scaffold component significantly improved the mechanical integrity of hydrogel component in the hybrid construct for weight-bearing applications. These results have collectively indicated that the manipulation of micro-architecture of scaffold could be an effective means to further regulate and guide desired cellular response in hybrid constructs.

Vascularization in bone tissue engineering constructs.

Vascularization of large bone grafts is one of the main challenges of bone tissue engineering (BTE), and has held back the clinical translation of engineered bone constructs for two decades so far. The ultimate goal of vascularized BTE constructs is to provide a bone environment rich in functional vascular networks to achieve efficient osseointegration and accelerate restoration of function after implantation. To attain both structural and vascular integration of the grafts, a large number of biomaterials, cells, and biological cues have been evaluated. This review will present biological considerations for bone function restoration, contemporary approaches for clinical salvage of large bone defects and their limitations, state-of-the-art research on the development of vascularized bone constructs, and perspectives on evaluating and implementing novel BTE grafts in clinical practice. Success will depend on achieving full graft integration at multiple hierarchical levels, both between the individual graft components as well as between the implanted constructs and their surrounding host tissues. The paradigm of vascularized tissue constructs could not only revolutionize the progress of BTE, but could also be readily applied to other fields in regenerative medicine for the development of new innovative vascularized tissue designs.

Three-dimensional fabrication of cell-laden biodegradable poly(ethylene glycol-co-depsipeptide) hydrogels by visible light stereolithography.

Stereolithography (SLA) holds great promise in fabrication of cell-laden hydrogels with biomimetic complexity for use in tissue engineering and pharmaceutics. However, the availability of biodegradable photocrosslinkable hydrogel polymers for SLA is very limited. In this study, a water-soluble methacrylated poly(ethylene glycol-co-depsipeptide) was synthesized to yield a biodegradable photocrosslinkable macromer for SLA. Structural analysis confirmed the inclusion of biodegradable peptide and ester groups and photocrosslinkable methacrylate groups into the polymer backbone. The new macromer combined with RGDS peptide was used for SLA fabrication of hydrogels in absence and presence of cells. With the increasing light exposure time in SLA, mechanical stiffness of the hydrogels increased from 3 ± 1 kPa to 38 ± 13 kPa. Total mass loss of the samples within 7 days in PBS was 13%-21% and within 24 days 35%-66%. Due to degradation, the mechanical stiffness decreased by one order magnitude within 7-day incubation in PBS. Encapsulated endothelial cells proliferated in the hydrogels during 10-day in vitro cell culturing study. The macromer was further used in SLA to fabricate bifurcating tubular structures as preliminary vessel grafts. The new biodegradable, photocrosslinkable polymer is a significant addition to the very limited material selection currently available for SLA-based fabrication of cell-laden tissue engineering constructs.

Porous calcium polyphosphate bone substitutes: additive manufacturing versus conventional gravity sinter processing-effect on structure and mechanical properties.

Porous calcium polyphosphate (CPP) structures proposed as bone-substitute implants and made by sintering CPP powders to form bending test samples of approximately 35 vol % porosity were machined from preformed blocks made either by additive manufacturing (AM) or conventional gravity sintering (CS) methods and the structure and mechanical characteristics of samples so made were compared. AM-made samples displayed higher bending strengths (≈1.2-1.4 times greater than CS-made samples), whereas elastic constant (i.e., effective elastic modulus of the porous structures) that is determined by material elastic modulus and structural geometry of the samples was ≈1.9-2.3 times greater for AM-made samples. X-ray diffraction analysis showed that samples made by either method displayed the same crystal structure forming ß-CPP after sinter annealing. The material elastic modulus, E, determined using nanoindentation tests also showed the same value for both sample types (i.e., E ≈ 64 GPa). Examination of the porous structures indicated that significantly larger sinter necks resulted in the AM-made samples which presumably resulted in the higher mechanical properties. The development of mechanical properties was attributed to the different sinter anneal procedures required to make 35 vol % porous samples by the two methods. A primary objective of the present study, in addition to reporting on bending strength and sample stiffness (elastic constant) characteristics, was to determine why the two processes resulted in the observed mechanical property differences for samples of equivalent volume percentage of porosity. An understanding of the fundamental reason(s) for the observed effect is considered important for developing improved processes for preparation of porous CPP implants as bone substitutes for use in high load-bearing skeletal sites.

Solid freeform fabrication of porous calcium polyphosphate structures for bone substitute applications: in vivo studies.

Porous calcium polyphosphate (CPP) structures with 30 volume percent porosity and made by solid freeform fabrication (SFF) were implanted in rabbit femoral condyle sites for 6-wk periods. Two forms of SFF implants with different stacked layer orientation were made in view of prior studies reporting on anisotropic/orthotropic mechanical properties of structures so formed. In addition, porous CPP implants of equal volume percent porosity made by conventional sintering and machining methods were prepared. Bone ingrowth and in vivo degradation of the three different implant types were compared using back-scattered scanning electron microscopy (BS-SEM) of implant samples and quantitative analysis of the images. The results indicated bone ingrowth with all samples resulting in 30-40% fill of available porosity by bone within the 6-wk period. In the 6-wk in vivo period, approximately 7-9% loss of CPP by degradation had occurred.

Characterizations of additive manufactured porous titanium implants.

This article describes physical, chemical, and mechanical characterizations of porous titanium implants made by an additive manufacturing method to gain insight into the correlation of process parameters and final physical properties of implants used in orthopedics. For the manufacturing chain, the powder metallurgy technology was combined with the additive manufacturing to fabricate the porous structure from the pure tanium powder. A 3D printing machine was employed in this study to produce porous bar samples. A number of physical parameters such as titanium powder size, polyvinyl alcohol (PVA) amount, sintering temperature and time were investigated to control the mechanical properties and porosity of the structures. The produced samples were characterized through porosity and shrinkage measurements, mechanical compression test and scanning electron microscopy (SEM). The results showed a level of porosity in the samples in the range of 31-43%, which is within the range of the porosity of the cancelluous bone and approaches the range of the porosity of the cortical bone. The results of the mechanical test showed that the compressive strength is in the wide range of 56-509 MPa implying the effect of the process parameters on the mechanical strengths. This technique of manufacturing of Ti porous structures demonstrated a low level of shrinkage with the shrinkage percentage ranging from 1.5 to 5%.

Mechanical characteristics of solid-freeform-fabricated porous calcium polyphosphate structures with oriented stacked layers.

This study addresses the mechanical properties of calcium polyphosphate (CPP) structures formed by stacked layers using a powder-based solid freeform fabrication (SFF) technique. The mechanical properties of the 35% porous structures were characterized by uniaxial compression testing for compressive strength determination and diametral compression testing to determine tensile strength. Fracture cleavage surfaces were analyzed using scanning electron microscopy. The effects of the fabrication process on the microarchitecture of the CPP samples were also investigated. Results suggest that the orientation of the stacked layers has a substantial influence on the mechanical behavior of the SFF-made CPP samples. The samples with layers stacked parallel to the mechanical compressive load are 48% stronger than those with the layers stacked perpendicular to the load. However, the samples with different stacking orientations are not significantly different in tensile strength. The observed anisotropic mechanical properties were analyzed based on the physical microstructural properties of the CPP structures.

Solid freeform fabrication and characterization of porous calcium polyphosphate structures for tissue engineering purposes.

Solid freeform fabrication (SFF) enables the fabrication of anatomically shaped porous components required for formation of tissue engineered implants. This article reports on the characterization of a three-dimensional-printing method, as a powder-based SFF technique, to create reproducible porous structures composed of calcium polyphosphate (CPP). CPP powder of 75-150 microm was mixed with 10 wt % polyvinyl alcohol (PVA) polymeric binder, and used in the SFF machine with appropriate settings for powder mesh size. The PVA binder was eliminated during the annealing procedure used to sinter the CPP particles. The porous SFF fabricated components were characterized using scanning electron microscopy, micro-CT scanning, X-ray diffraction, and mercury intrusion porosimetry. In addition, mechanical testing was conducted to determine the compressive strength of the CPP cylinders. The 35 vol % porous structures displayed compressive strength on average of 33.86 MPa, a value 57% higher than CPP of equivalent volume percent porosity made through conventional gravity sintering. Dimensional deviation and shrinkage analysis was conducted to identify anisotropic factors required for dimensional compensation during SFF sample formation and subsequent sintering. Cell culture studies showed that the substrate supported cartilage formation in vitro, which was integrated with the top surface of the porous CPP similar to that observed when chondrocytes were grown on CPP formed by conventional gravity sintering methods as determined histologically and biochemically.