**1. Introduction**

As reported in the U.S., 7.9 million fractures occur annually, of which 5–10% develop non-unions and/or delayed unions, which are major sources of complications in the treatment of bone fractures [1]. In 2005, 17 billion dollars in medical costs were attributed to the treatment of fractures caused by osteoporosis alone. By 2025, costs are estimated to rise to 25 billion dollars [2,3]. These numbers highlight the importance of achieving early mechanical stability and load-bearing capability in long weight-bearing bones. For this reason, the successful treatment of bone defects is of great importance. Vascularity and mechanical stability need to be taken into account. The "gold standard" of many surgical techniques used to reconstruct bone for critical-sized bone defects is the use of autologous bone tissue [4]. However, the use of autografts has limitations, like donor-side morbidity, additional operations, or limited availability of tissue, as well as geometric mismatch between the harvested bone and the defect site, which can result in voids and poor integration [5,6]. Further alternative substitutes are allografts and xenografts [7]. Xenograft (animal-derived material) approaches often carry risks, like inflammation and rejection of

**Citation:** Ahlhelm, M.; Latorre, S.H.; Mayr, H.O.; Storch, C.; Freytag, C.; Werner, D.; Schwarzer-Fischer, E.; Seidenstücker, M. Mechanically Stable β-TCP Structural Hybrid Scaffolds for Potential Bone Replacement. *J. Compos. Sci.* **2021**, *5*, 281. https://doi.org/10.3390/ jcs5100281

Academic Editors: Corrado Piconi and Simone Sprio

Received: 25 August 2021 Accepted: 9 October 2021 Published: 17 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the transplant due to physiological incompatibility of animal organs in human beings [8]. Therefore, research activity concerning bone-grafting approaches has shifted from natural grafts to synthetic bone graft substitutes and the use of biological factors [9]. Among these materials, next to metals (e.g., titanium, titanium alloys [10]) and bioglasses [11], ceramics like calcium phosphates (CaP; e.g., tricalcium phosphates (TCP), hydroxyapatite (HAp)) as well as added active growth factor recombinant human bone morphological proteins (e.g., rhBMPs) are the typical materials of choice, either alone or in combination [9,12]. Calcium phosphate ceramics are among the most commonly used and effective synthetic bone replacement materials. For example, β-tricalcium phosphate (β-TCP) is osteoconductive and is integrated into the bone without a disturbing connective tissue layer [13,14]. This property combined with its cell-mediated resorption enables the complete regeneration of bone defects. Pores and especially micropores (0.1 to 10 μm) promote bone ingrowth and can give β-TCP osteoinductive properties [13,15–18]. From this, it can be deduced that the bone implants should have similar properties, in terms of porosity, strength and stiffness, to the piece of bone to be replaced. If the strength of the implant is too low, there is a risk that the component will fail after implantation. However, if the strength and stiffness of the implant is too high, the surrounding bone will degrade. This process is known as "stress-shielding" and, like component failure, should be avoided at all costs [19,20]. The compressive strength of spongy bone is between 2–20 MPa, depending on the literature reference. With regard to porosity, cancellous bone has values between 50–90%, which explains the low mechanical load capacity. At the same time, however, the bone becomes light and the pores enable the supply of nutrients to the bone and the removal of metabolic products [21,22]. The majority of studies on CaP scaffolds focus on bone growth in the macropores (>100 μm), where bone structures such as osteones and trabeculae can form. However, more and more studies show that micropores (<50 μm) also play an important role. Not only do they improve bone growth in the macropores, but they also provide additional space for bone growth [23]. Bone growth in the micropores offers great mechanical advantages in CaP scaffolds, as it optimizes the properties of otherwise brittle materials by further stabilizing the implant and improving load transfer.

To date, specific material combinations have been examined in order to combine the tissue engineering advantages of organic materials with the mechanical load resistance of inorganic materials. Examples of such composite biomaterials are given in [24] and [25]. The latter reports on inorganic-organic hybrid scaffolds. Polyethylene glycol (PEG) and star poly(dimethylsiloxane) were mixed with bone-like matrices collagen type I, CaP, and osteocalcin, indicating that developed hybrid gels may prove promising for osteochondral regeneration. However, the compressive strength was limited by these polymers. In addition, no specific 3D construct was achievable. Ref. [24] reports on the fabrication of porous SF/β-TCP hybrid scaffolds for bone tissue reconstruction by a freeze-drying process. The manufactured scaffolds demonstrated high porosity (>60%) with good interpore connectivity and showed good biocompatibility. However, compressive strength and modulus were relatively low (<1 MPa), and no complex 3D scaffold was achieved.

Before the development of additive manufacturing technologies, ceramic bone replacement structures were usually manufactured using so-called dip coating processes in order to be able to approximately reproduce the filigree and highly porous structures [26]. Foams made of polyurethane, for example, serve as a lost form in this process. The polymer foam is cut to size and dipped into a ceramic slurry, which penetrates the pores. In a subsequent processing step, the foam mold is then burned out, and the scaffold is sintered. Although this approach can be used to produce highly porous structures, the resulting geometry is not greatly influenced but is rather predetermined by the (PU)foam. This disadvantage is overcome by the use of specific direct foaming or additive manufacturing technologies, since the mold geometry can be specifically modeled or is not needed at all. An example of an additively manufactured SiO2- and zinc-doped β-TCP scaffold is given in [27]. Although the results indicate that addition of dopants to the TCP scaffolds enhanced early stages of bone formation and implant fixation when compared to pure

TCP alone in a rabbit tibia model, the compressive strength of the achieved scaffolds only amounted to around 6 MPa.

The human bone consists of a dense and solid outer shell (*Substantia corticalis*) and an inner porous filling (*Substantia spongiosa*). In order to be able to reproduce such bone architectures with different structures, which could be used as implants in the future, two technologies were recently intelligently combined. The outer shell of the bone was produced using a commercial three-dimensional (3D) printer, and the sponge-like inner bone structure was reproduced by a ceramic foam [28,29]. For the foam production, socalled Freeze Foaming was used. In this approach, in a freeze dryer, the ambient pressure around an aqueous ceramic suspension is lowered, causing the suspension to first foam and then to suddenly freeze. Ongoing pressure reduction lets the frozen water sublimate, i.e., it evaporates without becoming liquid beforehand. A subsequent heat treatment produces a solid ceramic foam. In the next step, the porous bone-like structures are fitted to a customized, complex outer ceramic shell and, thereby, made mechanically more stable. This is where additive manufacturing (AM) comes into play. One of the best-known processes in AM is the conventional stereolithography (SLA) process. This process basically allows photopolymerizable suspensions, which are filled with ceramic particles, to be cured by a UV laser. Today, the commercially available material portfolio using lithography-based ceramic manufacturing (LCM) for high-performance components also works with β-TCP, thus playing a role in this contribution. The LCM technology as a projection-based (PSL) top-down process with a light source in the blue range (452–465 nm) is representative of the so-called ceramic additive manufacturing vat photopolymerization (CerAM VPP) process (Ceramic Additive Manufacturing Vat Photopolymerization). This allows a digital micro-mirror unit, which splits a light beam into individual pixels and then projects a digital image pixel-by-pixel onto the building platform. This makes it possible to image the entire contour of the component cross-section without a mask. Thus, layer by layer, a complex 3D structure is created. In a last hybridization step, the two methods can be combined to produce porous-dense, graded, structural hybrids by a joint sintering process. However, it is possible to not only foam within additively manufactured structures but also to foam them in. This solution makes it possible to provide a porous and sponge-like scaffold as the lead structure for cells to grow into, and at the same time, AM parts serve as load-bearing support structures. In this current study, advanced scaffolds made of β-TCP were manufactured and analyzed in terms of their biocompatibility in vitro and in vivo and tested for their mechanical behavior. The authors postulated that such a complex inorganic hybrid structure, due to the combination of load-bearing support and porous cell-ingrowthallowing interior, will eventually allow the manufacturing of bone-like mechanically stable implants that are potentially applicable for long-bone defects.
