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This blog was inspired by my own clinical experience with head and neck cancer patients who received extensive facial bone resection and subsequent reconstruction. These patients often have to suffer both the wrath of cancer as well as the disfiguring but lifesaving surgery. Even, when the surgeons are world-class experts, their options to help these patients with restoration are limited. The autologous bone graft is simply not enough. What many people perhaps don’t know is that bone harvesting is not only limited by donor sites and but also adding additional risk to surgeries.
Therefore, it is very exciting to see a myriad of research and commercial actives focusing on bone tissue engineering, especially ones focusing on 3D bioprinting bone (and cartilage). In fact, if we can truly design and manufacture implantable living bone tissue at ease, there is a huge economy behind the “space-filling” industry, from dentistry, reconstructive surgery, to orthopedics. [1-5]
That said, just because a 3D printed structure looks like bone, it may be far from the kind of bone we have in our bodies. Thus, this blog intends to address the different players in the bone bioprinting arena at a high level and interested readers can refer to the reference section of this blog below and a follow-up blog on recommended readings on the same subject.
Bone is a constantly changing dynamic organ that is no different in complexity than any other organ system we are working to regenerate. In addition to its structural and mechanical properties, the musculoskeletal system is an organ with both hard and soft tissues, vasculatures, different cellular components that are responsive to complex hormonal stimuli. In addition to structural support, bone also serves as a mineral deposit and site of hematopoiesis for the human body. 
There are also many different kinds of bones, some weight-bearing, and others not. Even a single piece of bone has different components, for example, trabecular versus cortical bone, which has entirely different mechanical and cellular properties. Therefore, bioprinting bone in a sense is no different than bioprinting any other organ system, except on the contrary to challenges faced in creating a replacement kidney, small pieces of bone graft (3d printed or not) have been in both research and commercial spaces for decades. 
That said, achieving successful bone bioprinting will need the following components (and possibly more) to work well together:
- Architectural design
- Bioprinter/3D printing process
- Cell biology
- Growth factor/Chemical cues for cell differentiation and growth
Several key concepts in bone tissue engineering need to be clarified before diving deeper. An ideal bone replacement will be both osteoinductive and osteoconductive.  The end goal is to achieve osteointegration. Osteoinduction is the process by which osteogenesis is induced. It is a phenomenon regularly seen in the healing of a fracture, with the recruitment of immature cells and the stimulation of these cells to develop into preosteoclasts. Osteoconduction means that bone grows along a surface, which is seen in the case of bone implants. Typically, materials with a rougher surface show better Osteoconduction. Osseointegration is the stable anchorage of an implant achieved by direct bone-to-implant contact. 
3D Printing Process for Bone Fabrication
Since bone fabrication is not a new field of research, what makes additive manufacturing an attractive modality for tissue engineers include the following: 1) Controlling macro- and microstructures of the scaffold to incorporate cells, drugs, proteins, and more. In other words, complete controlling of a structural and compositional complex product that few other tissue engineering techniques currently provide. 2) Producing patient-specific geometries. 3) New design possibilities of implants. For example, variation in scaffold porosity and density. 4) On-demand point-of-care production of needed bone graft. An ideal clinical workflow schema is suggested in Figure 1. Even direct cell bioprinting in situ has also been suggested. 
The paper “Integrating three-dimensional printing and nanotechnology for musculoskeletal regeneration” by Margaret Nowicki  listed in the reference section of this blog provides a summary of current major 3D printing processes for bone bio fabrication, including a limited discussion on pros and cons of the techniques. That said, a discussion of 3D bioprinters will not be complete without a discussion of material science, cell management, and chemistry, which are all critical to creating a favorable bone growth microenvironment.
The common fabrication techniques include:
- Inkjet Bioprinting
- Selective Laser Sintering
- Fused Deposition Modeling
- Electrospinning (not really 3D printing, but additive in nature)
Biomaterials for Bone Fabrication
An ideal bone replacement will be both osteoinductive and osteoconductive.  The end goal is to achieve osteointegration.
A lot of efforts have been put into researching biomaterials in bone bio fabrication/bioprinting. As Nowicki et al. point out, ideal scaffolds should present with “favorable hydrophilicity, roughness, and surface topography, at the micron and sub-micron level, to replicate the natural environment of native tissue. Scaffolds must present Nanoscale features on the surface topography of a scaffold increase surface area, surface-to-volume ration, and surface roughness enhancing cell adhesion and promoting favorable biocompatibility”.  The composition, architecture, and properties of biomaterial can be used to regulate the microenvironment in which cells reside and enhance osteogenesis.  Additional features that would favor a biomaterial include radio-opacity and biodegradability.
Some of the major biomaterials for bone fabrication include inorganic materials such as calcium phosphate (CaP) bioceramics, hydroxyapatite, PCL (polycaprolactone), and organic natural or synthetic biopolymers such as collagen or silk. The material choice tends to be based on a reductionist’s approach of mimicking the organic-inorganic composition of the native bone. Many of the said materials demonstrate osteoinduction and osteoconduction, promoting cell differentiation and favoring bone growth. Of these materials, bioceramics appear the most promising.  According to clinicaltrials.gov there are over 300 clinical trials being conducted in the world testing the effectiveness of calcium phosphate materials for bone applications, with 60 already completed in the U.S. alone, and an additional 58 in Europe. 
Early Successes and Startups
Early successes with 3D printed bone and cartilage graft are demonstrated primarily in animal models, but occasionally in human subjects (mostly in craniofacial reconstruction cases).
Through past 3DHEALS events and Pitch3D activities, there are several notable startups focusing on 3D bioprinting for bone and cartilage components. For example, Osteopore, a Singapore-based startup that demonstrated osteointegration in patients with calvaria burr holes using a 3D printed biodegradable mesh (“Osteoplug”) based on PCL material.  The company is also the manufacturer behind a recent well-publicized case with an Australian patient who received extensive bone graft and reconstruction for a bone defect as large as 36 centimeters.  Particle 3D is a new Danish startup focusing on developing patient-specific 3D printed biodegradable bone implants using calcium phosphate and fatty acid biomaterials with its proprietary 3d printing process. Particle 3D has demonstrated early successes in animal trials. Dimension Inx, a startup based in Chicago USA, focuses its products around the “Hyperelastic bone”, which is a 3D-printed synthetic scaffold, “composed of 90% by weight hydroxyapatite and 10% by weight poly(lactic-co-glycolic acid)”, which has demonstrated successful bone regeneration in an animal trial. Finally, Nanochon is a startup focusing on 3D bioprinting load-bearing implant based on nanostructured polyurethane (nPU) to replace damaged or lost cartilage, which does not have self-healing ability as bone does. Successful cartilage formation was observed in large animal models.
Challenges, and Thus Opportunities:
First of all, size matters. While bone has self-healing property, hence why hair-line fractures heal themselves without intervention other than immobility, larger bone defects are a challenge to fix even with existing bone graft solutions. Most of the published animal or human data only showed the early success of bone growth in defects in millimeter-scale. However, the author is looking forward to official publication related to a recent Australian case where a young man received bone scaffold for a much larger bone defect up to 36 centimeters.  Scalable production of replacement bone will determine the ultimate impact of such technology.
Secondly, many of the current solutions lack the range of mechanical properties required to replace or improve existing permanent implantation solutions. In particular, load-bearing 3D bio-printed bone or cartilage is still not available to replace metal-based implants.
Finally, solving the vascularization challenge will remain critical to successful bone regeneration as it is for regenerating any other organ system. Ongoing research strategies include synthesizing bioceramics with the vasculogenic ability and designing scaffold pore size and density favoring neovascularization. [3,5]
While challenges ahead, 3D bioprinting bone will perhaps be one of the most exciting fields in regenerative medicine in our immediate future. As opposed to bioprinting a functional kidney, small pieces of functional patient-specific 3D printed bone graft seem more readily available to the patients. Perhaps this will be the first killer app for the organogenesis industry.
- The potential impact of bone tissue engineering in the clinic. Mishra R1, et al Regen Med. 2016 Sep;11(6):571-87. doi: 10.2217/rme-2016-0042. Epub 2016 Aug 23.
- Use of Osteoplug polycaprolactone implants as novel burr-hole covers. Low S W, et al. Singapore Med J. 2009; 50(8) : 777
- Biomaterials for Craniofacial Bone Regeneration. Thrivikraman G, et al Dent Clin North Am. 2017 Oct;61(4):835-856. doi: 10.1016/j.cden.2017.06.003.
- Three-Dimensionally Printed Hyperelastic Bone Scaffolds Accelerate Bone Regeneration in Critical-Size Calvarial Bone Defects.Huang YH1, et al. Plast Reconstr Surg. 2019 May;143(5):1397-1407. doi: 10.1097/PRS.0000000000005530.
- Integrating three-dimensional printing and nanotechnology for musculoskeletal regeneration. Margaret Nowicki. Nanotechnology. 2017 September 20; 28(38): 382001. doi:10.1088/1361-6528/aa8351.
- Simple additive manufacturing of an osteoconductive ceramic using suspension melt extrusion. Slots C, et al. Dent Mater. 2017 Feb;33(2):198-208.