Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone

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Biological and Medical Applications of Materials and Interfaces

3D-Printed Polylactic Acid (PLA) Scaffolds Promote Bone-like Matrix Deposition In-vitro Rayan Fairag, Derek Rosenzweig, Jose Luis Ramirez Garcialuna, Michael H. Weber, and Lisbet Haglund ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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ACS Applied Materials & Interfaces

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3D-Printed Polylactic Acid (PLA) Scaffolds Promote

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Bone-like Matrix Deposition In-vitro

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Rayan Fairag , Derek H. Rosenzweig , Jose L. Ramirez-Garcialuna , Michael H. Weber and Lisbet Haglund

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Montreal, Canada. Orthopaedic Department, Faculty of Medicine, King Abdulaziz University,

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Jeddah, Saudi Arabia. Experimental Surgery, Department of Surgery, McGill University,

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Montreal, Canada. McGill Scoliosis and Spine Research Group. Shriners Hospital for Children,

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Montreal, Canada.

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Orthopaedic Research Laboratory, Division of Orthopaedic Surgery, McGill University, 2

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KEYWORDS: 3D printing; low-cost; human osteoblasts; mesenchymal stem cells; bone defect;

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PLA; scaffolds; bone repair; tissue engineering.

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ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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COVER ART: Desktop 3D-printer generating high-resolution PLA structures mimicking native

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tissues. PLA scaffolds, with different pore sizes (500 µm, 750 µm and 1000 µm), were fabricated.

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Scaffolds were cell seeded with primary human osteoblasts and compared for cell growth, activity,

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and bone-like tissue formation, in which 750 µm pore size scaffolds showed superiority over the

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other sizes. Further experiments confirmed the ability in supporting osteogenic differentiation of

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human MSC on 750 µm pore scaffolds. These findings suggest that low-cost 750 µm pores-size

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3D printed scaffolds may be suitable as a bone substitute for repair of bone defects.

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ABSTRACT

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Large bone defects represent a significant challenge for clinicians and surgeons. Tissue

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engineering for bone regeneration represents an innovative solution for this dilemma and may

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yield attractive alternate bone substitutes. 3D printing with inexpensive desktop printers show

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promise in generating high-resolution structures mimicking native tissues using biocompatible,

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biodegradable and cost-effective thermoplastics, which are already FDA approved for food use,

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drug delivery and many medical devices. Micro-porous 3D-printed PLA scaffolds, with different

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pore sizes (500 µm, 750 µm and 1000 µm), were designed and manufactured using an inexpensive

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desktop 3D-printer and mechanical properties were assessed. Scaffolds were compared for cell

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growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts

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showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which

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750 µm pore size scaffolds showed superiority. Further experimentation using human

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mesenchymal stem cells (MSC) on 750 µm pore scaffolds showed their ability in supporting

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osteogenic differentiation. These findings suggest that even in the absence of any surface

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modifications, low-cost 750 µm pores-size 3D printed scaffolds may be suitable as a bone

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substitute for repair of large bone defects.

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1. Introduction

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Critical size bone defects resulting from trauma, infections, degenerative diseases, tumor

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resections and other conditions such as non-union can be challenging for reconstructive

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orthopaedic surgery . In most cases, bone-grafting is required to fill the bone defects and to

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stimulate bone-healing of the affected area . Various methods of grafting have been established,

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and autologous bone grafting is considered the “gold standard” due to its biological advantages

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and cost-effectiveness . Despite its widespread use, autologous grafting exhibits some

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disadvantages and limitations such as invasiveness, graft site morbidity/pain, limited quantity,

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surgical wound infection, and increased operative time or multiple operations . Other grafting

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options include allograft and xenograft, which are less commonly used due to cost, potential

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disease transmission and immune rejection . To circumvent these issues, clinical bone cements

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have been developed to fill, stabilize and even promote repair of critical bone defects. Calcium

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phosphate bone cement is a synthetic graft substitute with osteoconductive and antibiotic delivery

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capabilities . However, they lack the mechanical properties required to withstand the applied

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loads. Poly(methyl methacrylate) (PMMA) cements provide strong mechanical support, yet they

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lack bone regenerative properties and are associated with potential drawbacks such as toxicity,

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thermal necrosis and damage to surrounding healthy tissue . Also, some bone cements have been

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shown to cause local stress around the target area which can lead to secondary fractures . Hard

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metals, such titanium, possess superior mechanical properties. Nonetheless, they come with a high

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cost, issues with bone integration, tissue inflammation and dehiscence .

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Tissue engineering approaches for bone repair and regeneration have been under investigation

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to circumvent many of the issues described above. This approach combines cells,

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biomaterial/scaffolds and bioactive factors to create a construct that is able to adjust to the

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physiological status and regenerate lost tissue . Among the types of scaffolds used for bone tissue

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engineering, three-dimensional (3D) printing has become an attractive approach. 3D printing (also

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known as additive manufacturing) is an advanced technology of designing and fabricating 3D

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structures with high precision, accuracy and detailed biomimetics in a rapid fashion. This

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technology can overcome many limitations of conventional fabrication options such as solvent-

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casting, fiber meshes and gas foaming . Current devices for 3D printing can generate scaffolds

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ranging from millimeter to nanometer scales . Advantages of using 3D printing include the

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capability to create versatile scaffolds using different FDA-approved synthetic biodegradable

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polymers which have been termed Generally Recognized as Safe (GRAS) polymers. These

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polymers include poly lactic acid (PLA) , polycaprolactone (PCL) and polyurethanes (PUs) which

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have been used for food packaging, drug delivery, and medical and surgical devices .

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The ideal material for scaffold development should fulfill specific criteria. The material must

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be biocompatible and must be capable of being generated with an interconnected network to mimic

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the natural tissue architecture . The scaffolds should provide a microenvironment promoting cell

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attachment, growth/ingrowth, and differentiation toward the desired lineage . Scaffolds should

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also provide the optimal conditions to form functional tissue. It must allow fabrication in different

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and irregular complex shapes and not induce toxicity or inflammation . Finally, 3D printed

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scaffolds should possess suitable mechanical properties , appropriate porosity and pore size, and

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they should be cost effective . Studies have shown that scaffolds with pore size ranging between

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100 µm to 1500 µm are suitable for tissue engineering applications including bone . Furthermore,

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it’s been suggested that larger pore size (>500 um) stimulates and conducts vascular tissue growth

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which is necessary for bone healing

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Among the common thermoplastics for 3D printing, PLA is a biodegradable, highly versatile,

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aliphatic polyester that can be derived fully from natural renewable resources. It has drawn much

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attention and has been investigated in biomedical and surgical fields, such as surgical sutures and

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operating tools, orthopaedic screws and fixation materials, drug carriers and tissue engineering

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scaffolds

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were suitable for chondrocyte and nucleus pulposus tissue engineering applications . In this study,

. We have previously demonstrated that low-cost desktop 3D printed PLA plastics

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we tested the feasibility of using low-cost 3D printed PLA scaffolds of three different pore sizes

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(500 µm, 750 µm and 1000 µm) on primary human osteoblast adhesion, growth and osteogenic

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matrix deposition. Furthermore, we evaluated the ability of human mesenchymal stem cells

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(MSCs) to adhere, proliferate, and differentiate toward osteogenic phenotype and form mineralized

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clusters on scaffolds with a specific pore size. These low-cost 3D-printed scaffolds present a

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promising candidate for bone defect repair and graft substitution.

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2. Materials and Methods 2.1.

Fabrication of the materials

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PLA Raptor Series filament (FDA approved as GRAS and autoclavable) was purchased from

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MakerGeeks (Springfield, MO USA) with (1.75 mm) thickness. 3D, uniform, open-pore cuboidal

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scaffolds with square pore sizes of 500 µm (small), 750 µm (medium) and 1000 µm (large) were

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designed using CAD Software SolidWorks® (Dassault Systems SolidWorks Corporation,

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Waltham, MA) and converted to stereolithography (.stl) file format (Figure 1A). The overall

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dimensions of the scaffolds were 10 mm × 10 mm × 4 mm. The .stl files were formatted to meet

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printing parameters on the Flashforge Creator Pro 3D Desktop Printer (Flashforge, Los Angeles,

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USA). PLA filament extrudes at an optimal temperature of 210 °C from a 0.3 mm nozzle. Printing

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time was consistent with 50 minutes to print small pore size, 45 minutes for printing medium pore

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size and 39 minutes for large pore size scaffolds. Constructs were then rinsed in 70% ethanol

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overnight, packaged and autoclaved using standard dry cycle (121 C, 20 psi for 30 minutes).

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2.2.

Scaffold characterization (μCT)

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Micro-CT analysis of empty PLA scaffolds was performed using a Skyscan 1172 instrument

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(Bruker, Kontich, Belgium) at a channel resolution of 7 microns using NRecon v.1.6.10.4 to

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compose 3D models and CTAn v.1.16.4.1 software for quantitative analysis (Bruker) as described


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previously . Quantitative data for PLA scaffolds was recorded in a region of interest (ROI) 10 mm

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wide x 10 mm long x 4 mm in depth spanning the entirety of the scaffold. Reported parameters

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are scaffold volume / ROI volume (SV/RV %), scaffold surface (SS mm ), number of scaffold

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filaments per mm (SF.N 1/mm), average thickness of the scaffold filament (SF.Th µm), average

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space between filaments or pore size (SF.Sp µm), total porosity (Po.Tot %).

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2.3.

Mechanical testing

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All scaffold dimensions were measured after printing to ensure dimensional consistency. Failing

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point protocol applied un-confined vertical axial compression to scaffold surfaces at a rate of 0.1

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mm/s until heights were reduced by 50% (1 mm) using a Mini Bionix 858 (MTS machine). The

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material stiffness was obtained by calculating the Young’s modulus between 5% - 10%

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compressive strain for all scaffolds as previously reported . 30

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2.4.

Cell Isolation and culture (Osteoblasts and MSCs)

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2.4.1. Osteoblasts:

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Human lumbar spine tissues were obtained from 3 donors (aged 13, 17 and 52) with institutional

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ethical approval and with donor family consent in collaboration with the local organ donation

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program (Transplant Quebec). Cortical bone was cut manually into small 1-2 mm pieces and

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washed vigorously with phosphate buffer saline (PBS) supplemented with antibiotics. All visible

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muscle and fascia were removed by a sterile scalpel. To remove any remaining soft tissue, the

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pieces were incubated overnight in growth medium (high-glucose DMEM; 0.1 mM nonessential

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amino acids; 10 mM HEPES; 1 mM sodium pyruvate; 10% fetal bovine serum; and 1%

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gentamycin solution supplemented with 1.5 mg/mL collagenase type II (Invitrogen/Gibco,

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Burlington, ON, Canada). The remaining bone was washed 3 times with sterile PBS and placed in


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a T75 flask covered with growth media (7 days) and osteoblastic cells were obtained by outgrowth

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from the bone pieces, trypsinized and subcultured for 1 passage.

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2.4.2. Bone Marrow Mesenchymal Stem cells:

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Human bone marrow-derived MSC from a 20-year-old female, were obtained from RoosterBio

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Inc., Frederick, MD 21703, USA. Cells were tested and certified by the manufacturer. Cells were

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expanded for 1 passage following manufacturer’s protocol using (hMSC media booster GTX-

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SU003 and hMSC high-performance basal media SU005). Each independent experiment with

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MSCs was performed at passage 2 in this study.

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2.5.

Scaffold seeding and Cell attachment

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Osteoblasts and MSC were cultured in monolayer (High glucose-Dulbecco’s Modified Eagle

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Medium (DMEM) containing 10% (v/v) fetal bovine serum (Gibco, Burlington, ON, Canada), 1%

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glutaMAX supplement and 0.5% gentamycin) at 37 °C in a humidified cell culture incubator with

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5% CO2. Confluent cells (passage 2) were washed with sterile PBS, detached using 0.25% trypsin

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(Gibco, Burlington, ON, Canada). Fresh culture media was added, and cells were centrifuged at

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500 × g for five minutes. Detached cells were suspended in fresh culture media and seeded on the

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scaffolds, using a syringe technique developed “in-house”. Briefly, scaffolds were placed inside

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5mL syringes fitted with a four-way stopcock with rotating collar (Navilyst medical Inc.,

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Marlborough, MA USA) with a cell density of 5x10 cells/scaffold. Syringes were kept inside the

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culture incubator and rotated every 30 minutes over 2 hours to give equal opportunity for cell

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adhesion. After that, constructs were transferred to 24 well-plate (with remaining cell suspension)

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overnight. Next morning, scaffolds were transferred to a new dish, and immersed with osteogenic

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media (DMEM low glucose, 10 % FBS, 1 % gentamycin, 50 μg/ml ascorbic acid, 10 nM

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dexamethasone, 5 mM betaglycerol-2-phosphate) or standard differentiation media (DMEM high

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glucose, 10 % FBS, 1 % gentamycin) for 7, 14 or 21 days.

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2.6.

Cell number and DNA Quantification

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After 21 days of culture, scaffolds were submerged in a fixed volume (1 mL) of 4M Guanidine

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hydrochloride (GuHCl) buffer supplemented with complete protease inhibitor cocktail (Roche

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Applied Science, Indianapolis, IN, USA). The DNA content of 3D constructs was measured using

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DNA HOECHST 33258 Assay. Briefly, all (GuHCl) extracts were diluted 10-fold before analysis.

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Calf-thymus DNA supplemented with an equivalent amount of 0.4 M GuHCl was used to generate

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standard curves

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prepared according to manufacturer instructions (ThermoFisher). Samples were analyzed in 96-

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well microplates, in triplicate, using a Tecan M200 Pro plate reader (Tecan, Mannedorf,

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Switzerland), at 360 nm excitation and 460 nm emission, and 420 nm cut-off. Standard curves

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were generated on the worksheet. We quantified the DNA content of 5 x 10 cells and thereby

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extrapolated DNA count to 7 pg DNA/cell.

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. The Hoechst 33258 (Molecular Probes, ThermoFisher, Burlington, ON) was

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2.7.

Protein analysis and matrix production (Western blot)

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200 µl of the protein extracts were ethanol precipitated and re-suspended in NuPage LDS Loading

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Buffer (Life Technologies) as described previously . Total protein in the extracts was determined

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by Pierce™ Coomassie (Bradford) Protein Assay Kit (ThermoFisher Scientific Inc., Burlington,

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ON, Canada). Proteins were separated on 10% LDS polyacrylamide gels and electro-transferred

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to Amersham proton 0.2 nitrocellulose membrane (Hybond-P, GE Healthcare, Mississauga,

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Canada). Membranes were blocked with 3% skim milk and probed with antibodies against human

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Osteopontin (OPN) (R&D Systems, Minneapolis, MN, USA) at 1:1000 dilution. Appropriate

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secondary antibody (R&D Systems, Minneapolis, MN, USA) at 1:1000 dilution was used. Alpha-

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tubulin (Abcam, 1:5000) was used as a loading control. All membranes were incubated with the

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appropriate secondary antibodies and developed using Western Lightning Plus-ECL (PerkinElmer

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Life Sciences) and LAS 4000 Image Quant system (General Electric). Densitometric calculations

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was carried out using ImageLab software (Biorad). Samples were normalized to Alpha-tubulin and

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to the background of the blot.

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2.8.

Calcified matrix deposition (Alizarin Red staining)

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After 21 days culture period, PLA scaffolds of all sizes (Small, Medium and Large) seeded with

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osteoblasts, MSCs or acellular constructs ( standard culture (STD) and osteogenic (OST) ) were

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fixed with 4% buffered paraformaldehyde solution at room temperature for 10 min. Scaffolds were

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then washed three times with PBS to remove formalin, and 1% Alizarin Red solution (Sigma-

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Aldrich Inc., Darmstadt, Germany) was added and left for 10 minutes at room temperature. All

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samples were washed 2-4 times with distilled water until dye is removed from the acellular

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negative controls. Images were captured with a Canon EOS 350d digital camera . Alizarin red

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stain was dissolved and subsequently quantified using the osteogenesis assay kit (ECM815

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Millipore Sigma Inc., Canada) according to the user manual.

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2.9.

Osteogenesis related gene expression

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500 µg RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the

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manufacturer instructions, following 21 days of culture. RNA was reverse-transcribed using

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qScript reagent (Quantabio, Beverly, MA USA). Quantitative real-time RT-PCR analysis was

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performed with StepOnePlus, Real Time PCR System (Applied Biosystems, Foster City, CA,

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USA) using PerfeCTa SYBR Green detection reagent (Quantabio, Beverly, MA USA). Relative

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expression of Runt-related transcription factors (RUNX2), alkaline phosphatase (ALP), bone

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sialoprotein (BSP), osteonectin (ON) and collagen type I (COL-1) was quantified and normalized

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to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative

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expression and fold change for each target gene was evaluated using 2

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(Table 1) were previously validated and described

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Table 1. Sequence of the forward and reverse primers used for the q-PCR.

-ΔΔCt

method . All primer sets 34

.

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Target gene

Forward primer (5'-3')

Reserved primer (5'-3')

GAPDH

TCCCTGAGCTGAACGGGAAG

GGAGGAGTGGGTGTCGCTGT

BSP

AAGCTCCAGCCTGGGATGA

TATTGCACCTTCCTGAGTTGAACT

ON

TCCGTACGGCAGCCACTAC

GCATGGCTCTCAAGCACTTG

RUNX-2

TCAGCCCAGAACTGAGAAACTC

TTATCACAGATGGTCCCTAATGGT

ALP

AGAACCCCAAAGGCTTCTTC

CTTGGCTTTTCCTTCATGGT

Col1a1

AGGGCTCCAACGAGATCGAGATCCG

TACAGGAAGCAGACAGGGCCAACG

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2.10.

Scanning electron microscopy observation

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Constructs were assessed by scanning electron microscopy (FEI Inspect F50 FE-SEM) after 21

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days of culture. Briefly, cell seeded and acellular scaffolds were fixed for 30 minutes with 4%

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paraformaldehyde, rinsed with PBS and subsequently underwent critical point drying process as


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previously described , followed by Platinum sputter coating. Morphological characteristics of the

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constructs and attached cells were observed using SEM operating at 5 kV high resolution.

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2.11.

Statistical analysis

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All values are expressed as mean ± standard deviation and represent at least three independent

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experiments (n = 3). For all comparisons of effects, simple t-test or one-way ANOVA tests were

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performed. Multiple comparisons were adjusted using Tukey post-hoc tests where appropriate. P-

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values < 0.05 - < 0.0001 were considered significant. GraphPad Prism version 6.00 was used for

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all statistical analyses (GraphPad Inc., La Jolla, CA).

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3. Results

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3.1.

Characterization of scaffolds (Morphology)

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The scaffold printing process generated a well-defined architecture with uniform pore distribution

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(Figure 1A). The orthogonal structure was recorded with the size of 10x10x4 mm and surface

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morphology of the constructs was demonstrated grossly and with µCT. The dry weight of each

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scaffold was highly consistent with the amount of material filling and composing the scaffold

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(small: 0.36 gm ± 0.002, Medium: 0.23 gm ± 0.002, Large: 0.24 gm ± 0.004) p < 0.0001 (Figure

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1B). All three scaffold types had measured pore sizes harmonious with the intended designs,

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suggesting accuracy of the low-cost desktop printer. The pore sizes were highly consistent for each

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scaffold type and suitable for cells to invade from the top as well as from side walls. The small

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pore scaffolds had average pore size of 585.61 µm ± 26.40, the medium had average pore size of

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769.94 µm ± 12.98, large size scaffolds had average pore size of 1028.85 µm ± 57.54, p < 0.0001.

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Scaffolds were designed with porosity volumes allowing better nutrient and cell interaction (Small:

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45.82 % ± 10.43, medium: 66.10 % ± 5.45, large: 83.9 % ± 4.95), p < 0.005 (Figure 1C). The

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scaffold fabrication and replication process manifests high accuracy and precision as evidenced by


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µCT analysis which proves the value of low-cost printing in tissue engineering applications.

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SV/RV for small scaffolds had an average of 54.17 % ± 10.43, the medium had an average of

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33.89 % ± 5.45, and the large scaffolds had an average of 29.99 % ± 2.87) p < 0.05. Average

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scaffold surface area for the small scaffold was 630.87 mm ± 18.93, the medium size average

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surface area was 489.28 mm ± 21.51, and the large size average surface area was 371.63 mm ±

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8.64, p < 0.0001. The average filament/mm for small scaffolds was 0.94 ± 0.04, for the medium

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0.62 ± 0.04 and for the large scaffold 0.64 ± 0.01. p < 0.0001. Thickness of filament was consistent

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in all sizes with no statistical difference observed (Small: 0.46 mm ± 0.03, medium: 0.46 mm ±

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0.01, large: 0.43 mm ± 0.03) (Table 2).

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Figure 1. Morphological characterization of 3D printed scaffolds. A) Representative images of

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the 3D models with dimensions and printing process. B) Quantification of scaffold weight, (n =

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6), error bars represent ± SD (** = P value < 0.005), (# = P value < 0.0001), with a representative


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image of printed scaffolds (Canon EOS 350d Camera). C) Pore size was calculated by Scanning

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Electron Microscopy, and porosity was determined by µ-CT. For each set, (n = 3), error bars

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represent ± SD and (* = P value < 0.05), (** = P value < 0.005), (# = P value < 0.0001).

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Table 2. µCT Characterization of 3D-printed PLA scaffolds Variable

Small pore size

Medium pore size

Large pore size

SV/RV (%)

54.2 ± 10.4

33.9 ± 5.4

29.9 ± 2.8

SS (mm )

630.9 ± 18.9

489.2 ± 21.5

371.6 ± 8.6

SF.N (1/mm)

0.94 ± 0.04

0.64 ± 0.04

0.62 ± 0.01

SF.Th (µm)

460.5 ± 34.9

467.4 ± 17.2

439.3 ± 35.7

SF.Sp (µm)

577.1 ± 66.0

798.9 ± 93.9

1,005 ± 42.4

Po.Tot (%)

45.8 ± 7.4

66.1 ± 5.4

83.9 ± 4.9

2

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3.2.

Mechanical properties:

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The compressive mechanical characteristics of the PLA scaffolds are presented in (Figure 2).

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Significant differences in stiffness was observed between the three sizes (p < 0.05, p < 0.0001) in

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which the Young’s Modulus for the small pore size was (206.7 MPa ± 0.17 SD), medium size


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scaffold (137.5 MPa ± 6.98 SD) and (116.4 MPa ± 5.97 SD) for large size PLA scaffold (Figure

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2A). The failure point of each scaffold was determined from the stress/strain curves in which the

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small size failure point was around (21.63 MPa), around (11.86 MPa) for the medium size and

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around (8.53 MPa) for the large pore scaffold (Figure 2B). Our results demonstrated an overall

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higher compressive modulus with smaller pores due to the addition of bulk material (smallest pore

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size has the highest amount of material and is the stiffest). These scaffolds show compressive

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properties comparable to trabecular bone when strained between 5% and 10% and the determined

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Young’s Modulus values were comparable to the wide range of measurements in the literature . 39

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Figure 2. Mechanical properties of 3D printed scaffolds. A) Young’s modulus representing 5-10%

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compressive stress/strain curves of printed PLA scaffolds. For each set, (n = 3), error bars represent

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± SD and (* = P value < 0.05), (# = P value < 0.0001). B) Stress/Strain curves of (500 µm, 750

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µm and 1000µm) showing the amount of deformation, Elastic (proportionality) limit and plastic

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region. For each set, (n = 3).

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3.3.

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3.3.1. Osteoblasts

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3.3.1.1.

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Cell behavior and activity within the 3D environment

3D cell organization and growth in scaffolds

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Cell growth and spatial distribution was visualized during the culture period using an inverted light

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microscope (Axiovert 40, Göttingen, Germany). Osteoblasts were surrounding and attaching to

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the surface of the scaffold and growing into the open pores in all three scaffold sizes. An additional

286

observation found during the culture period was that the cell network always starts at the edges of

287

the pores. With longer culture time, there was an extension of cell growing into the center of the

288

pore, especially the corner pores of the scaffolds (Figure 3). Cells were also growing and covering

289

the surface and between the additive layers of the scaffolds indicating cell adaptation to the 3D

290

environment (Figure 4A).

291


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292

Figure 3. Scanning electron microscopy of acellular and cell-seeded scaffolds. Representative

293

SEM images of (acellular, Osteoblasts, MSC-OST seeded scaffolds) at 80x, 450x, 1500x and

294

22000x magnifications and scale bars represents 1 mm, 200 µm, 50 µm and 5 µm with rectangular

295

marker indicating the region of scan (n = 3).

296 297

3.3.1.2.

Osteoblast doubling and expansion

298

Assessment of cell population expansion over the culture period was evaluated by measuring total

299

DNA content. All scaffolds showed an increase in osteoblast number with the highest number of

300

osteoblasts recorded from medium scaffolds. Statistically significant differences were

301

demonstrated supporting the superiority of the medium size p < 0.0001. Scaffolds with small pore

302

size had approximately (683,437 ± 27,455) cells, scaffolds with medium pore size had around

303

(765,945 ± 27,455) and scaffolds with large size had (612,519 ± 24,438) cells (Figure 4B).

304

3.3.1.3.

Calcification and mineralization

305

Calcium deposition and mineralization was qualitatively assessed by alizarin red staining with

306

empty scaffold cultured the same period served as control. Osteoblast scaffolds showed intense

307

alizarin red staining in all sizes with the medium size scaffold displaying the strongest color

308

intensity. Quantification of alizarin red concentrations (mM) showed significantly more calcified

309

matrix (P < 0.05) from the medium size scaffold compared to the other sizes. The small scaffold

310

showed (1.44 ± 0.034 mM concentration), the medium scaffolds showed (2.48 ± 0.167 mM

311

concentration), and the large showed (1.030 ± 0.102 mM concentration) (Figure 4C).

312

3.3.1.4.

Assessment of osteogenic matrix


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313

The expression of OPN, a major extracellular bone matrix protein, was evaluated by western blot

314

analysis of the cell-scaffold extracts seeded with primary osteoblasts. Scaffolds showed

315

significantly high expression of OPN with the medium pore size scaffold showing the highest

316

amount (Small: 4.41 ± 0.94, Medium: 6.76 ± 0.64, Large: 2.04 ± 0.24) p < 0.0005 (Figure 4D).

317 318

Figure 4. Primary human osteoblast culture on 3D printed scaffolds. A) Representative images of

319

Bright field microscopy images (phase contrast) of osteoblast-seeded scaffolds during culture

320

period at 10× magnification (n = 6). Black arrows indicate cell growth and neo-tissue deposition.

321

Scale bar represents 500 µm. B) DNA quantification of osteoblast/scaffold using HOECHST

322

33258 Assay. Horizontal line in the middle of the graph represents 5 x 10 cells. (n = 3) Error bars 5


18

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323

represent ± SD and (** = P value < 0.005), (*** = P value < 0.0005), (# = P value < 0.0001). C)

324

Representative image of fixed acellular and osteoblast-seeded PLA scaffolds of (500 µm, 750 µm

325

and 1000 µm) pore size stained with Alizarin Red-S stain after 21 days culture (n = 3). Marker

326

represent 10x10 mm squares dimensions and stain quantification using Alizarin Red osteogenesis

327

kit (n = 3) Error bars represent ± SD and (** = P value < 0.005), (*** = P value < 0.0005). D)

328

Representative Western blot for Osteopontin (OPN) accumulation and densitometry quantification

329

(n = 3) Error bars represent ± SD, (* = P value < 0.05), (*** = P value < 0.0005).

330 331

3.3.2. Bone marrow stem cells:

332

3.3.2.1.

3D cell organization and growth in scaffolds

333

During culture period, MSCs started to form colonies within the pores creating nanofibril lines.

334

Cells had a strong tendency to form multilayers, coating the scaffold with matrix-like tissue that

335

proliferated deep into the pore spaces with time (Figure 5A). This was confirmed by scanning

336

electron microscopy (SEM) which showed abundant network formation covering the scaffolds

337

with densely packed coating at the surface. This matrix filled the pores over time. The high

338

proliferation rates and cellular activity indicates biocompatibility of our 3D printed PLA scaffolds

339

(Figure 3).

340

3.3.2.2.

Population and expansion of MSCs

341

MSCs showed higher activity than osteoblasts during experiments, indicated by requirement of

342

more frequent media change. MSCs showed comparable growth on the medium scaffold and no

343

statistical difference in number between MSCs cultured in standard and osteogenic media was

344

observed. Approximately (728,424 ± 40,140) MSC-STD and (790,108 ± 13,287) MSC-OST


19


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345

(Figure 5B). These results were also comparable to osteoblast seeded on medium size scaffold (no

346

statistical difference observed).

347

3.3.2.3.

Calcified matrix deposition on scaffolds

348

Scaffolds were seeded with undifferentiated MSCs and were cultured in the presence or absence osteogenic

349

differentiation media. Calcium deposition and mineralization was qualitatively assessed by alizarin red

350

staining with cultures in standard and osteogenic media, and empty scaffolds served as a negative control.

351

Scaffolds cultured in both standard and osteogenic media showed evenly distributed cells after 21 days of

352

culture. Alizarin red staining was assessed and showed significantly values (p < 0.0001) in osteogenic

353

compared to standard cultures supporting calcified matrix deposition, (MSC-STD: 0.054 ± 0.002 mM

354

concentration), (MSC-OST:3.667 ± 0.376 mM concentration) (Figure 5C). In fact, comparing quantified

355

calcified matrix of MSC-OST to osteoblast on medium scaffold showed significantly more calcified matrix

356

in the MSC cultures (p < 0.05).

357

3.3.2.4.

Assessment of MSC osteogenesis

358

Differentiation of human bone marrow MSCs on medium pore size PLA scaffold toward

359

osteogenic phenotype was assessed by the expression of osteogenic gene markers RUNX2, ALP,

360

BSP, ON and COL-1 at 7, 14 and 21 days of culture. Cells displayed significantly higher

361

expression of BSP (89.181 ± 2.385-fold difference, p < 0.0001) ALP (18.070 ± 0.951-fold

362

difference, p < 0.05) RUNX-2 (3.184 ± 0.159-fold difference, p < 0.05) where COL-1 and ON

363

showed non-statistically significant elevation compared to the standard group after 21 days.

364

(Figure 5D). Furthermore, MSCs supplemented with osteogenic differentiation media showed

365

abundant OPN protein expression compared to cultures in standard media (Figure 5E).

366

Densitometry quantification showed a significant difference and supported these findings (MSC-


20

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367

STD: 2.06 ± 0.34, MSC-OST: 5.11 ± 1.45, p < 0.001). Together, these data support the

368

differentiation of MSCs seeded on these PLA 3D-printed scaffolds towards osteogenic phenotype.

369 370

Figure 5. Human MSCs culture on medium sized scaffolds. A) Representative images of Bright

371

field microscopy (phase contrast) of MSC-seeded scaffolds (osteogenic vs standard culture) during

372

culture period at 10× magnification (n = 6). Black arrows indicate cell growth and neo-tissue

373

deposition. Scale bar represents 500 µm. B) DNA quantification of MSC/scaffold (osteogenic vs

374

standard culture) using HOECHST 33258. Horizontal line in the middle of the graph represents

375

500 000 cells. (n = 3) Error bars represent ± SD. C) Fixed acellular, and MSC-seeded PLA

376

scaffolds of (osteogenic vs standard culture) stained with Alizarin Red-S stain after 21 days

377

culture. Representative images (n = 3) of Alizarin Red-S staining with marker representing 10x10


21


Page 22 of 43

378

mm squares Error bars represent ± SD, (# = P value < 0.0001). D) qPCR analysis of osteogenic

379

markers after 21 days culture. Expression levels were normalized to GAPDH with fold change

380

compared to standard culture media (n = 3) Error bars represent ± SD, (* = P value < 0.05) (# = P

381

value < 0.0001). E) Representative Western blot for Osteopontin (OPN) accumulation and

382

densitometry quantification (n = 3) Error bars represent ± SD, (** = P value < 0.005).

383 384

4. Discussion

385

PLA is a FDA-approved Generally Recognized as Safe (GRAS) polymer used for many

386

biomedical applications with excellent biocompatibility, biodegradability, and mechanical

387

properties . Our PLA constructs demonstrated high accuracy and reproducibility of fabrication.

388

All Scaffolds were designed with same outer dimensions. The amount of material needed to print

389

the medium and large scaffolds is similar to preserve dimensional precision, but a larger amount

390

of material is needed for the small pore size scaffold. The number, size and distribution of pores

391

and strut size varies between the designs. Moreover, the results from this study clearly demonstrate

392

that a narrow difference in pore size (500 – 750 – 1000 µm) significantly impacts the mechanical

393

properties, cell growth, differentiation and calcified matrix production. Our data help to narrow

394

the optimal wide range of appropriate pore size for bone regeneration described in the literature

395

(between 100-1500 µm). The presented scaffolds were designed with a porosity volume (40-80%)

396

that keeps optimal balance between mechanical strength and degradation rate . Our findings were

397

similar to what has been recently described in other studies using additive manufacturing strategies

398

in regard to appropriate pore size and porosity for optimal osteoconduction

399

concept, primary human osteoblasts were used to show biocompatibility of these scaffolds

400

following the 3D-printing process and to determine the appropriate pore size for cells to grow,

26

40

. As a proof of

41-43


22

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401

differentiate or maintain their phenotype. Here, the 750 µm PLA scaffold showed optimal cell

402

growth and activity, as demonstrated by the highest calcified neo-matrix deposition and high

403

expression of OPN compared to the other two sizes. Bone marrow MSCs were then used to

404

determine the feasibility of osteogenic differentiation within the 750 µm pore-size scaffold. We

405

showed that 3D-Printed PLA scaffolds with 750 µm pore size supported proliferation and

406

osteogenic differentiation of MSCs, as indicated by significant production calcified bone-like

407

matrix, OPN and osteogenic gene expression. Cell-seeding of the scaffolds was sufficient without

408

any type of coating or cell carrier. We took into consideration that some cells fail to attach, and

409

the cell numbers in the scaffolds after 3 weeks is relative to the starting number of adherent cells.

410

Moreover, our study revealed that the 3D-printed PLA scaffolds presented here have

411

mechanical properties that fall within the range reported for native bone

412

mechanical properties between models and materials depend on geometric microstructure, and

413

factors such as printing technique, print settings (material flow rate, rate of printing) and the

414

percentage polymers in raw materials. Also, the type of testing conducted can play a major role in

415

the mechanical characterization of the scaffold. Our data indicate that if implanted into a bone

416

defect, these scaffolds may provide an appropriate environment to recruit host stem cells and

417

promote osteogenic differentiation and bone repair. In vivo studies will be necessary to determine

418

potential adverse effects, bone repair and scaffold resorption rates.

419

. Differences in

44-46

It comes without surprise that 3D-printing has been strongly adopted by orthopaedic surgery

420

clinical practice, medical education, patient education

421

While 3D printing has been used for some time to generate patient models of defects for pre-

422

surgical planning , there is a growing shift in using this technology in actual bone or tissue repair.

423

One major focus in orthopaedic and reconstructive surgery is to use 3D-printed constructs for

47-49

and orthopaedic-related basic science

.

50-52

53


23


Page 24 of 43

424

filling bone defects, substituting current standard therapies as an innovative approach for bone

425

repair

426

of 3D-printed polymers as graft substitute. Hung and colleagues used a custom 3D printer to create

427

a hybrid scaffold composed of PCL combined with decellularized bone particles from calves in-

428

vitro using human adipose stem cells and implanted these cell-seeded scaffolds in a calvarial defect

429

animal model. The authors showed their model’s effectiveness toward stimulating bone

430

regeneration and also highlighted that using a hybrid-type scaffold could affect mechanical

431

properties of the scaffold

432

(PLA/hydroxyapatite) composite printed with advanced mini-deposition system with a pore size

433

of 500 µm and 60% porosity, and seeded them with rat stem cells . Another group has recently

434

shown the clinical potential of using silk/recombinant human BMP-2 composite scaffolds using

435

techniques in which 3D printing could be a feasible option to use . Zigang and his group evaluated

436

in vitro behavior of osteoblast cell-lines within 3D powder printed Poly (lactic-co-glycolic acid)

437

PLGA scaffolds, highlighting their feasibility for bone regeneration applications. The authors

438

described the limitations of using established cell lines versus primary extracted cells . Not many

439

studies have applied a low-cost commercial desktop 3D printer which can be a part of many

440

laboratories, handled by everyone with countless research applications and benefits. These devices

441

typically require minimal physical space and take minutes to hours to print the desired model

442

depending on multiple factors under the control of the consumer.

. Several studies have shown applicability and clinical relevance of using different types

54-56

. Zhang and colleagues showed similar results using a

46

57

58

40

443

Due to the expiration of key patents in the past decade, high quality and highly precise desktop

444

3D printers can be purchased for under $1000 USD . A major advantage of low-cost 3D-printing

445

is that most of the thermoplastic materials such as (PLA, PLGA, PCL), ceramics (calcium

446

polyphosphate) and metal (titanium) have met FDA medical devices, biologics and drug

12


24

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447

regulations as safe polymers (GRAS), bringing the clinical translation of these tools within reach.

448

To increase the level of promise of this low-cost technology, a group of clinicians fabricated

449

surgical retractors using PLA fabrics which have been approved by the FDA for surgical use. The

450

devices exhibited safety, cost effectiveness and sufficient mechanical strength generated using a

451

single low-cost 3D printer (The Replicator 2, ~ $2,000 USD) . This supports the idea that these

452

technologies can be employed to produce surgical instruments at remote or under-developed sites

453

at a very low cost. Furthermore, Ayoub and colleagues fabricated custom drill guides using FDA

454

approved materials for dental implant surgery using an inexpensive printer, demonstrating an

455

advantage in lowering postoperative morbidity rates and operation times .

59

60

456

Another area of rapid growth in 3D printing is in the emerging field bioprinting. A vast range

457

of natural raw materials are available such as alginate, chitosan, gelatin, collagen and hyaluronic

458

acids can be used as “bioinks” in low-cost bioprinters . Contributing to its relatively affordable

459

costs in which some units are available between $ 5000 - $10000 USD, native/natural materials

460

and hardware can easily be modified to allow for tremendous progress in 3D-printing process.

461

Bioprinters affirmed their importance and role in developing new tissue engineering approaches ,

462

advance cancer research models , pharmacological screening, and therapeutic agent delivery .

463

Additionally, vascularization of 3D-printed constructs is now possible using bioprinting

464

technology .

61

62

63

64

65

465

Most studies are suggesting that 3D-printed bone graft substitutes are ready for and require

466

additional studies beyond animal models. A limited number of clinical studies composed mainly

467

of case reports and small-size population studies can be found using 3D-printed models as implants

468

66-67

469

outcomes .

. Indeed, 3D-printed titanium hip implants have been used for over 10 years, with positive patient 68


25


Page 26 of 43

470

To consider these low-cost 3D printed scaffolds suitable for use in patients, many factors must

471

be taken into account. Perhaps most surgeons would consider biomechanics as a major limiting

472

factor in the clinical use of this technology. It is important to note that our PLA in vitro model

473

lacks surface modifications which may enhance cell adhesion, cell differentiation or bone matrix

474

deposition. Since we do not perform surface modifications, the only factors affecting cell

475

proliferation and matrix production were likely due to the nature of flat 3D-printed PLA, pore size

476

differences, structural geometry, surface roughness and chemistry.

477

While we may argue that these scaffolds work well as a bone substitute in vitro, certain

478

modifications can enhance the properties of PLA. Chemical modification with minerals such as

479

hydroxyapatite or tri-calcium phosphate can be mixed and added to PLA filaments to induce more

480

osteoinductivity, attract cells and promote bone repair . Soft hydrogels such as chitosan can be

481

incorporated with growth factors within the open space of scaffolds to provide a better proliferative

482

environment and conduct more osteogenesis . Physical and microstructural modifications can also

483

be used to adjust the scaffold mechanical/degradation rates.

69

70

484

5. Conclusion

485

The current study focused on emphasizing the value of inexpensive desktop 3D printers and

486

off-the-shelf materials in keeping pace with the advancement of tissue engineering. This study

487

demonstrates three different pore-sized 3D printed PLA scaffolds have been successfully

488

generated using an inexpensive desktop 3D printer. Out of three pore-sizes, the medium size (750

489

µm) scaffold exhibited highest cytocompatibility and bioactivity of primary human osteoblasts.

490

For clinical relevance, human MSCs seeded into the medium size scaffolds showed strong

491

osteogenic differentiation capacity. Therefore, we believe this simple, low-cost approach can be

492

extended to future in vivo studies with potential clinical applications. 3D-printed PLA could


26

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493

therefore be used as custom, on-site bolt or push fit into a bone defect allowing bone marrow stem

494

cells to infiltrate, adhere, proliferate and form new bone.

495 496 497

AUTHOR INFORMATION

498

Corresponding Author

499

Lisbet Haglund

500

McGill University, Department of Surgery, Montreal General Hospital, Room C10.148.2, 1650

501

Cedar Ave, Montreal, QC H3G 1A4, Telephone: (514) 934 1934 ext. 35380. OR Shriners Hospital

502

for Children,1003, boulevard Décarie, Montréal, Québec H4A 0A9, Telephone: (514) 282-7166,

503

[email protected]

504

Author Contributions

505

Conceptualization by R.F, D.H.R, M.H.W and L.H. Experimental designing by D.H.R and L.H.

506

Experimentation and data analysis carried out by R.F and D.H.R. Micro-CT analysis was done by

507

JLRGL Resources and Material were provided by L.H. Results interpretation by R.F, D.H.R and

508

L.H. Original draft was written by RF. Reviewing and editing by D.H.R and L.H. Project

509

Supervision by D.H.R, M.H.W and LH. All authors approved the final version of the manuscript

510

for submission.

511

Funding Sources

512

This research was supported by the Reseau de Recherche en Sante Buccodentaire et Osseuse

513

(RSBO) bone and oral health network (DHR, MHW and LH). Funding was also kindly provided


27


Page 28 of 43

514

by a Saudi Arabian Cultural Bureau Research Fellowship (RF). (JLRGL) was funded by the

515

Mexican National Council for Science and Technology.

516

Discloser Statement

517

No potential conflict of interest was reported by the authors.

518

ACKNOWLEDGMENT

519

Preparation of 3D printed samples for SEM and image acquisition was performed at the Materials

520

Engineering department at McGill University, Montreal QC, Canada.

521 522

ABBREVIATIONS

523

MSC, Mesenchymal stem cells; PLA, Poly lactic acid; PMMA, Poly(methyl methacrylate); PCL,

524

Polycaprolactone; PUs, polyurethanes; SV/RV, Scaffold volume / ROI volume; SS, scaffold

525

surface; SF.N, Number of scaffold filaments per mm; SF.Th, average thickness of the scaffold

526

filament; SF.Sp, Average space between filaments or pore size; Po.Tot, total porosity; PBS,

527

phosphate buffer saline; DMEM, High glucose-Dulbecco’s Modified Eagle Medium; FBS, Fetal

528

bovine serum; GuHCl, Guanidine hydrochloride; OPN, Osteopontin; STD, standard culture; OST,

529

osteogenic culture; RUNX2, Runt-related transcription factors; ALP, alkaline phosphatase; BSP,

530

bone sialoprotein; ON, osteonectin; COL-1, collagen type I; GAPDH, glyceraldehydes 3-

531

phosphate dehydrogenase; GRAS, FDA-approved Generally Recognized as Safe.

532 533

REFERENCES

534 535 536

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537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580


(3) De Boer, H. H. The history of bone grafts. Clinical orthopaedics and related research 1988, 226, 292-298. (4) Sen, M.; Miclau, T. Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? Injury 2007, 38 (1), S75-S80. (5) Dimitriou, R.; Jones, E.; McGonagle, D.; Giannoudis, P. V. Bone regeneration: current concepts and future directions. BMC medicine 2011, 9 (1), 66. (6) Dunne, N.; Clements, J.; Wang, J. Acrylic cements for bone fixation in joint replacement. In Joint Replacement Technology; Elsevier: 2014; pp 212-256. (7) Kim, D. H. Minimally Invasive Percutaneous Spinal Techniques E-Book, Elsevier Health Sciences: 2010. (8) Khor, E. From Academia to Entrepreneur: Lessons from the Real World, Elsevier: 2013. (9) Park, H. K.; Dujovny, M.; Agner, C.; Diaz, F. G. Biomechanical properties of calvarium prosthesis. Neurological research 2001, 23 (2-3), 267-276. (10) Vacanti, J. P.; Langer, R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. The lancet 1999, 354, S32-S34. (11) Peltola, S. M.; Melchels, F. P.; Grijpma, D. W.; Kellomäki, M. A review of rapid prototyping techniques for tissue engineering purposes. Annals of medicine 2008, 40 (4), 268-280. (12) Ventrici de Souza, J.; Liu, Y.; Wang, S.; Dorig, P.; Kuhl, T. L.; Frommer, J.; Liu, G. Y. ThreeDimensional Nanoprinting via Direct Delivery. The journal of physical chemistry. B 2018, 122 (2), 956-962, DOI: 10.1021/acs.jpcb.7b06978. (13) Schaschke, C.; Audic, J.-L., biodegradable materials. Multidisciplinary Digital Publishing Institute: 2014. (14) Compton, B. G.; Lewis, J. A. 3D Printing: 3D-Printing of Lightweight Cellular Composites (Adv. Mater. 34/2014). Advanced Materials 2014, 26 (34), 6043-6043. (15) O'brien, F. J. Biomaterials & scaffolds for tissue engineering. Materials today 2011, 14 (3), 88-95. (16) Studart, A. R. Additive manufacturing of biologically-inspired materials. Chemical Society Reviews 2016, 45 (2), 359-376. (17) Murphy, S. V.; Atala, A. 3D bioprinting of tissues and organs. Nature biotechnology 2014, 32 (8), 773. (18) Hulbert, S.; Young, F.; Mathews, R.; Klawitter, J.; Talbert, C.; Stelling, F. Potential of ceramic materials as permanently implantable skeletal prostheses. Journal of Biomedical Materials Research Part A 1970, 4 (3), 433-456. (19) Jones, A. C.; Arns, C. H.; Sheppard, A. P.; Hutmacher, D. W.; Milthorpe, B. K.; Knackstedt, M. A. Assessment of bone ingrowth into porous biomaterials using MICRO-CT. Biomaterials 2007, 28 (15), 2491-2504. (20) Murphy, C. M.; Haugh, M. G.; O'Brien, F. J. The effect of mean pore size on cell attachment, proliferation and migration in collagen–glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials 2010, 31 (3), 461-466. (21) Wake, M. C.; Patrick Jr, C. W.; Mikos, A. G. Pore morphology effects on the fibrovascular tissue growth in porous polymer substrates. Cell transplantation 1994, 3 (4), 339-343. (22) Sicchieri, L. G.; Crippa, G. E.; de Oliveira, P. T.; Beloti, M. M.; Rosa, A. L. Pore size regulates cell and tissue interactions with PLGA–CaP scaffolds used for bone engineering. Journal of tissue engineering and regenerative medicine 2012, 6 (2), 155-162.


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710 711 712 713


(70) Dong, L.; Wang, S. J.; Zhao, X. R.; Zhu, Y. F.; Yu, J. K. 3D- Printed Poly(epsilon-caprolactone) Scaffold Integrated with Cell-laden Chitosan Hydrogels for Bone Tissue Engineering. Sci Rep 2017, 7 (1), 13412, DOI: 10.1038/s41598-017-13838-7.


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Desktop 3D-printer generating high-resolution PLA structures mimicking native tissues. PLA scaffolds, with different pore sizes (500 µm, 750 µm and 1000 µm), were fabricated. Scaffolds were cell seeded with primary human osteoblasts and compared for cell growth, activity, and bone-like tissue formation, in which 750 µm pore size scaffolds showed superiority over the other sizes. Further experiments confirmed the ability in supporting osteogenic differentiation of human MSC on 750 µm pore scaffolds. These findings suggest that low-cost 750 µm pores-size 3D printed scaffolds may be suitable as a bone substitute for repair of bone defects.


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COVER ART: Desktop 3D-printer generating high-resolution PLA structures mimicking native tissues. PLA scaffolds, with different pore sizes (500 µm, 750 µm and 1000 µm), were fabricated. Scaffolds were cell seeded with primary human osteoblasts and compared for cell growth, activity, and bone-like tissue formation, in which 750 µm pore size scaffolds showed superiority over the other sizes. Further experiments confirmed the ability in supporting osteogenic differentiation of human MSC on 750 µm pore scaffolds. These findings suggest that low-cost 750 µm pores-size 3D printed scaffolds may be suitable as a bone substitute for repair of bone defects.



Figure 1. Morphological characterization of 3D printed scaffolds. A) Representative images of the 3D models with dimensions and printing process. B) Quantification of scaffold weight, (n = 6), error bars represent ± SD (** = P value < 0.005), (# = P value < 0.0001), with a representative image of printed scaffolds (Canon EOS 350d Camera). C) Pore size was calculated by Scanning Electron Microscopy, and porosity was determined by µ-CT. For each set, (n = 3), error bars represent ± SD and (* = P value < 0.05), (** = P value < 0.005), (# = P value < 0.0001).


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Figure 2. Mechanical properties of 3D printed scaffolds. A) Young’s modulus representing 5-10% compressive stress/strain curves of printed PLA scaffolds. For each set, (n = 3), error bars represent ± SD and (* = P value < 0.05), (# = P value < 0.0001). B) Stress/Strain curves of (500 µm, 750 µm and 1000µm) showing the amount of deformation, Elastic (proportionality) limit and plastic region. For each set, (n = 3).



Figure 3. Scanning electron microscopy of acellular and cell-seeded scaffolds. Representative SEM images of (acellular, Osteoblasts, MSC-OST seeded scaffolds) at 80x, 450x, 1500x and 22000x magnifications and scale bars represents 1 mm, 200 µm, 50 µm and 5 µm with rectangular marker indicating the region of scan (n = 3).


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Figure 4. Primary human osteoblast culture on 3D printed scaffolds. A) Representative images of Bright field microscopy images (phase contrast) of osteoblast-seeded scaffolds during culture period at 10× magnification (n = 6). Black arrows indicate cell growth and neo-tissue deposition. Scale bar represents 500 µm. B) DNA quantification of osteoblast/scaffold using HOECHST 33258 Assay. Horizontal line in the middle of the graph represents 5 x 10 cells. (n = 3) Error bars 5

represent ± SD and (** = P value < 0.005), (*** = P value < 0.0005), (# = P value < 0.0001). C) Representative image of fixed acellular and osteoblast-seeded PLA scaffolds of (500 µm, 750 µm and 1000 µm) pore size stained with Alizarin Red-S stain after 21 days culture (n = 3). Marker represent 10x10 mm squares dimensions and stain quantification using Alizarin Red osteogenesis



kit (n = 3) Error bars represent ± SD and (** = P value < 0.005), (*** = P value < 0.0005). D) Representative Western blot for Osteopontin (OPN) accumulation and densitometry quantification (n = 3) Error bars represent ± SD, (* = P value < 0.05), (*** = P value < 0.0005).


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Figure 5. Human MSCs culture on medium sized scaffolds. A) Representative images of Bright field microscopy (phase contrast) of MSC-seeded scaffolds (osteogenic vs standard culture) during culture period at 10× magnification (n = 6). Black arrows indicate cell growth and neo-tissue deposition. Scale bar represents 500 µm. B) DNA quantification of MSC/scaffold (osteogenic vs standard culture) using HOECHST 33258. Horizontal line in the middle of the graph represents 500 000 cells. (n = 3) Error bars represent ± SD. C) Fixed acellular, and MSC-seeded PLA scaffolds of (osteogenic vs standard culture) stained with Alizarin Red-S stain after 21 days culture. Representative images (n = 3) of Alizarin Red-S staining with marker representing 10x10 mm squares Error bars represent ± SD, (# = P value < 0.0001). D) qPCR analysis of osteogenic markers after 21 days culture. Expression levels were normalized to GAPDH with fold change



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compared to standard culture media (n = 3) Error bars represent ± SD, (* = P value < 0.05) (# = P value < 0.0001). E) Representative Western blot for Osteopontin (OPN) accumulation and densitometry quantification (n = 3) Error bars represent ± SD, (** = P value < 0.005).

Table 1. Sequence of the forward and reverse primers used for the q-PCR. Target gene

Forward primer (5'-3')

Reserved primer (5'-3')

hGAPDH

TCCCTGAGCTGAACGGGAAG

GGAGGAGTGGGTGTCGCTGT

BSP

AAGCTCCAGCCTGGGATGA

TATTGCACCTTCCTGAGTTGAACT

ON

TCCGTACGGCAGCCACTAC

GCATGGCTCTCAAGCACTTG

RUNX-2

TCAGCCCAGAACTGAGAAACTC

TTATCACAGATGGTCCCTAATGGT

ALP

AGAACCCCAAAGGCTTCTTC

CTTGGCTTTTCCTTCATGGT

hCol1a1

AGGGCTCCAACGAGATCGAGATCCG

TACAGGAAGCAGACAGGGCCAACG


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Table 2. µCT Characterization of 3D-printed PLA scaffolds Variable

Small pore size

Medium pore size

Large pore size

SV/RV (%)

54.2 ± 10.4

33.9 ± 5.4

29.9 ± 2.8

SS (mm )

630.9 ± 18.9

489.2 ± 21.5

371.6 ± 8.6

SF.N (1/mm)

0.94 ± 0.04

0.64 ± 0.04

0.62 ± 0.01

SF.Th (µm)

460.5 ± 34.9

467.4 ± 17.2

439.3 ± 35.7

SF.Sp (µm)

577.1 ± 66.0

798.9 ± 93.9

1,005 ± 42.4

Po.Tot (%)

45.8 ± 7.4

66.1 ± 5.4

83.9 ± 4.9

2


Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone

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