The extracellular matrix of bone can be pictured as a material made of parallel interspersed domains of fibrous piezoelectric collagenous materials and non-piezoelectric non-collagenous materials. To mimic this feature for enhanced bone regeneration, a material made of two parallel interspersed domains, with higher and lower piezoelectricity, respectively, is constructed to form microscale piezoelectric zones (MPZs). The MPZs are produced using a versatile and effective laser-irradiation technique in which K0.5Na0.5NbO3 (KNN) ceramics are selectively irradiated to achieve microzone phase transitions. The phase structure of the laser-irradiated microzones is changed from a mixture of orthorhombic and tetragonal phases (with higher piezoelectricity) to a tetragonal dominant phase (with lower piezoelectricity). The microzoned piezoelectricity distribution results in spatially specific surface charge distribution, enabling the MPZs to bear bone-like microscale electric cues. Hence, the MPZs induce osteogenic differentiation of stem cells in vitro and bone regeneration in vivo even without being seeded with stem cells. The concept of mimicking the spatially specific piezoelectricity in bone will facilitate future research on the rational design of tissue regenerative materials.

DNA-based biomaterials have been proposed for tissue engineering approaches due to their predictable assembly into complex morphologies and ease of functionalization. For bone tissue regeneration, the ability to bind Ca2+ and promote hydroxyapatite (HAP) growth along the DNA backbone combined with their degradation and release of extracellular phosphate, a known promoter of osteogenic differentiation, make DNA-based biomaterials unlike other currently used materials. However, their use as biodegradable scaffolds for bone repair remains scarce. Here, we describe the design and synthesis of DNA hydrogels, gels composed of DNA that swell in water, their interactions in vitro with the osteogenic cell lines MC3T3-E1 and mouse calvarial osteoblast, and their promotion of new bone formation in rat calvarial wounds. We found that DNA hydrogels can be readily synthesized at room temperature, and they promote HAP growth in vitro, as characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. Osteogenic cells remain viable when seeded on DNA hydrogels in vitro, as characterized by fluorescence microscopy. In vivo, DNA hydrogels promote the formation of new bone in rat calvarial critical size defects, as characterized by micro-computed tomography and histology. This study uses DNA hydrogels as a potential therapeutic biomaterial for regenerating lost bone.

This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

In the last three decades, all efforts in bone tissue engineering were driven by the dogma that the ideal pore size in bone substitutes lies between 0.3 and 0.5 mm in diameter. Newly developed additive manufacturing methodologies for ceramics facilitate the total control over pore size, pore distribution, bottleneck size, and bottleneck distribution. Therefore, this appears to be the method of choice with which to test the aforementioned characteristics of an ideal bone substitute. To this end, we produced a library of 15 scaffolds with diverse defined pore/bottleneck dimensions and distributions, tested them in vivo in a calvarial bone defect model in rabbits, and assessed the clinically most relevant parameters: defect bridging and bony regenerated area. Our in vivo data revealed that the ideal pore/bottleneck dimension for bone substitutes is in the range of 0.7-1.2 mm, and appears therefore to be twofold to fourfold more extended than previously thought. Pore/bottleneck dimensions of 1.5 and 1.7 mm perform significantly worse and appear unsuitable in bone substitutes. Thus, our results set the ideal range of pore/bottleneck dimensions and are likely to have a significant impact on the microarchitectural design of future bone substitutes for use in orthopedic, trauma, cranio-maxillofacial and oral surgery.

Guided bone regeneration (GBR) is the most common technique for localized bone augmentation.

The search for the perfect bone graft material is an important topic in material science and medicine. Despite human bone being the ideal material, due to its composition, morphology, and familiarity with cells, autografts are widely considered demanding and cause additional stress to the patient because of bone harvesting. However, human bone from tissue banks can be used to prepare materials in eligible form for transplantation. Without proteins and fats, the bone becomes a non-immunogenic matrix for human cells to repopulate in the place of implantation. To repair bone losses, the granulate form of the material is easy to apply and forms an interconnected porous structure. A granulate composed of β-tricalcium phosphate, pulverized human bone, and chitosan-a potent biopolymer applied in tissue engineering, regenerative medicine, and biotechnology-has been developed. A commercial encapsulator was used to obtain granulate, using chitosan gelation upon pH increase. The granulate has been proven in vitro to be non-cytotoxic, suitable for MG63 cell growth on its surface, and increasing alkaline phosphatase activity, an important biological marker of bone tissue growth. Moreover, the granulate is suitable for thermal sterilization without losing its form-increasing its convenience for application in surgery for guided bone regeneration in case of minor or non-load bearing voids in bone tissue.

The role of neutrophils in bone regeneration remains elusive. In this study, it is shown that intramuscular implantation of interleukin-8 (IL-8) (commonly recognized as a chemotactic cytokine for neutrophils) at different levels lead to outcomes resembling those of fracture hematoma at various stages. Ectopic endochondral ossification is induced by certain levels of IL-8, during which neutrophils are recruited to the implanted site and are N2-polarized, which then secrete stromal cell-derived factor-1α (SDF-1α) for bone mesenchymal stem cell (BMSC) chemotaxis via the SDF-1/CXCR4 (C-X-C motif chemokine receptor 4) axis and its downstream phosphatidylinositol 3'-kinase (PI3K)/Akt pathway and β-catenin-mediated migration. Neutrophils are pivotal for recruiting and orchestrating innate and adaptive immunocytes, as well as BMSCs at the initial stage of bone healing and regeneration. The results in this study delineate the mechanism of neutrophil-initiated bone regeneration and interaction between neutrophils and BMSCs, and innate and adaptive immunities. This work lays the foundation for research in the fields of bone regenerative therapy and biomaterial development, and might inspire further research into novel therapeutic options.

Mandibular osteoradionecrosis is a severe side effect of radiotherapy after the treatment of squamous cell carcinomas of the upper aerodigestive tract. As an alternative to its treatment by micro-anastomosed free-flaps, preclinical tissular engineering studies have been developed. Total bone marrow (TBM) associated with biphasic calcium phosphate (BCP) significantly enhanced bone formation in irradiated bone. One mechanism, explaining how bone marrow cells can help regenerate tissues like this, is the paracrine effect. The bone marrow cell extract (BMCE) makes use of this paracrine mechanism by keeping only the soluble factors such as growth factors and cytokines. It has provided significant results in repairing various tissues, but has not yet been studied in irradiated bone reconstruction. The purpose of this study was to evaluate the effect of BMCE via an intraosseous or intravenous delivery, with a calcium phosphate scaffold, in irradiated bone reconstruction. Twenty rats were irradiated on their hind limbs with a single 80-Gy dose. Three weeks later, surgery was performed to create osseous defects. The intraosseous group (n = 12) studied the effect of BMCE in situ, with six combinations (empty defect, BCP, TBM, BCP-TBM, lysate only, BCP-lysate). After four different combinations of implantation (empty defect, BCP, TBM, BCP-TBM), the intravenous group (n = 8) received four intravenous injections of BMCE for 2 weeks. Five weeks after implantation, samples were explanted for histological and scanning electron microscopy analysis. Lysate immunogenicity was studied with various mixed lymphocyte reactions. Intravenous injections of BMCE led to a significant new bone formation compared to the intraosseous group. The BCP-TBM mixture remained the most effective in the intraosseous group. However, intravenous injections were more effective, with TBM placed in the defect, with or without biomaterials. Histologically, highly cellularized bone marrow was observed in the defects after intravenous injections, and not after an in situ use of the lysate. The mixed lymphocyte reactions did not show any proliferation after 3, 5, or 7 days of lysate incubation with lymphocytes from another species. This study evaluated the role of BMCE in irradiated bone reconstruction. There were significant results arguing in favor of BMCE intravenous injections. This could open new perspectives to irradiated bone reconstruction.

Acid bone lysates (ABLs) represent the growth factors and other molecules released during autologous graft resorption. However, the impact of these bone-derived growth factors on the healing of bone defects has not yet been investigated. The aim of the present study was, therefore, to examine the impact of ABLs adsorbed to collagen membranes on bone regeneration. To this end, in 16 female Sprague Dawley rats, a standardized 5-mm-diameter critical size defect on the calvarial bone was created. The defects were covered with collagen membranes that had been soaked either in serum-free media or ABLs followed by lyophilization. After a healing period of 4 weeks, micro-computed tomography (μCT) and histological analyses by means of undecalcified thin ground sections were performed. μCT analysis of the inner 4 mm of the calvaria defect showed a greater bone defect coverage in the control group when compared to ABL group, 29.8% (confidence interval [CI]: 17.7-50.3) versus 5.6% (CI: 1.0-29.8, p = .03), respectively. Moreover, we found significantly more absolute bone volume (BV) in the control group when compared to ABL group, 0.59 mm3 (CI: 0.27-1.25) versus 0.07 mm3 (CI: 0.06-0.59, p = .04), respectively. Histomorphometry confirmed these findings with a relative BV in the central compartment of 14.1% (CI: 8.4-20.6) versus 5.6% (CI: 3.4-7.9, p = .004), respectively. These findings indicate that bone-derived growth factors contained in ABLs are able to attenuate bone regeneration within collagen membranes.

Endochondral bone development and regeneration relies on activation and proliferation of periosteum derived-cells (PDCs). Biglycan (Bgn), a small proteoglycan found in extracellular matrix, is known to be expressed in bone and cartilage, however little is known about its influence during bone development. Here we link biglycan with osteoblast maturation starting during embryonic development that later affects bone integrity and strength. Biglycan gene deletion reduced the inflammatory response after fracture, leading to impaired periosteal expansion and callus formation. Using a novel 3D scaffold with PDCs, we found that biglycan could be important for the cartilage phase preceding bone formation. The absence of biglycan led to accelerated bone development with high levels of osteopontin, which appeared to be detrimental to the structural integrity of the bone. Collectively, our study identifies biglycan as an influencing factor in PDCs activation during bone development and bone regeneration after fracture.

Angiogenin (ANG) is a potent stimulator of angiogenesis. The aim of this study was to fabricate an ANG-loaded scaffold and to evaluate its angiogenic and osteogenic effects. In this study, we fabricated an ANG-loaded scaffold using bovine bone powder and fibrin glue. We then evaluated the structural, morphological, and mechanical properties of the scaffold and the in vitro release profile of ANG. Cell proliferation, viability, and adhesion were evaluated using endothelial cells in vitro, and angiogenesis and new bone formation were evaluated using a rabbit calvarial defect model in vivo.

Resorbable synthetic and natural polymer-based membranes have been extensively studied for guided tissue regeneration. Alloplastic biomaterials are often used for tissue regeneration due to their lower immunoreactivity when compared with allogeneic and xenogeneic materials. Plenum® Guide is a synthetic membrane material based on polydioxanone (PDO), whose surface morphology closely mimics the extracellular matrix. In this study, Plenum® Guide was compared with collagen membranes as a barrier material for bone-tissue regeneration in terms of acute and subchronic systemic toxicity. Moreover, characterizations such as morphology, thermal analysis (Tm = 107.35 °C and crystallinity degree = 52.86 ± 2.97 %, final product), swelling (thickness: 0.25 mm ≅ 436% and 0.5 mm ≅ 425% within 24 h), and mechanical tests (E = 30.1 ± 6.25 MPa; σ = 3.92 ± 0.28 MPa; ε = 287.96 ± 34.68%, final product) were performed. The in vivo results revealed that the PDO membranes induced a slightly higher quantity of newly formed bone tissue than the control group (score: treated group = 15, control group = 13) without detectable systemic toxicity (clinical signs and evaluation of the membranes after necropsy did not result in differences between groups, i.e., non-reaction -> tissue-reaction index = 1.3), showing that these synthetic membranes have the essential characteristics for an effective tissue regeneration. Human adipose-derived stem cells (hASCs) were seeded on PDO membranes; results demonstrated efficient cell migration, adhesion, spread, and proliferation, such that there was a slightly better hASC osteogenic differentiation on PDO than on collagen membranes. Hence, Plenum® Guide membranes are a safe and efficient alternative for resorbable membranes for tissue regeneration.

Rationale: The effectiveness of stem cell based-therapy for bone regeneration has been demonstrated; yet, clinical application of autologous stem cells is still limited by invasive acquisition, long culture processes, and high cost. Besides, it remains controversial whether autologous stem cells could directly participate in tissue repair after differentiation. Thus, increasing allogeneic stem cells have been developed into drugs to indirectly activate endogenous regeneration and induce tissue regeneration. Human amniotic mesenchymal stromal cells (HAMSCs) have been extensively studied, showing multiple regulatory functions, but mechanisms of HAMSCs in promoting bone regeneration are remain unclear. Methods: Proteome profile of HAMSCs and their functions on vascularized bone regeneration were investigated in vitro, while rabbit cranial defect model was used to further detect the effects of bone formation in vivo. Results: HAMSCs secrete many osteogenic, angiogenic, and immunomodulatory cytokines. In vitro, HAMSCs can promote human bone-marrow mesenchymal stromal cells (HBMSCs) migration and osteogenic differentiation; promote the capillary-tube formation of human umbilical vascular endothelial cells (HUVECs), induce HUVECs migration and pro-angiogenic genes expression, and promote M2 macrophage polarization. Further, in vivo studies suggested that transplanted HAMSCs could survive and induce M2 macrophages to secrete bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) in rabbits' skull defects at an early stage, and, in turn, promote more new bone formation. Conclusion: HAMSCs have good biocompatibility and paracrine function to promote bone repair by stimulating endogenous regeneration.

Bone marrow stromal cells (BMSCs) are a rich source of osteogenic progenitor cells. A fundamental question is whether systemically transplanted BMSCs participate in bone regeneration. Luciferase and GFP double-labeled BMSCs were transplanted into irradiated mice. Five weeks after transplantation, artificial bone wounds were created in the mandibles and calvaria of the recipients. Animals were sacrificed at weeks 2, 4, and 6 after surgery and the expressions of luciferase and GFP were determined using Xenogen IVIS Imaging System, immunohistochemical staining and RT-PCR. The results demonstrated that transplanted BMSCs can be detected in wound sites as early as 2 weeks and lasted the whole experimental period. Luciferase expression peaked at 2 weeks after surgery and decreased thereafter, exhibiting a similar expression pattern as that of BSP, while GFP expression was relatively stable during the experimental period. In conclusion, BMSCs can migrate to bone wound sites and participate in bone regeneration in orocraniofacial region.

Decellularization is a promising new method to prepare natural matrices for tissue regeneration. Successful decellularization has been reported using various tissues including skin, tendon, and cartilage, though studies using hard tissue such as bone are lacking. In this study, we aimed to define the optimal experimental parameters to decellularize natural bone matrix using 0.5% sodium dodecyl sulfate and 0.1% NH4OH. Then, the effects of decellularized bone matrix on rat mesenchymal stem cell proliferation, osteogenic gene expression, and osteogenic differentiations in a two-dimensional culture system were investigated. Decellularized bone was also evaluated with regard to cytotoxicity, biochemical, and mechanical characteristics in vitro. Evidence of complete decellularization was shown through hematoxylin and eosin staining and DNA measurements. Decellularized bone matrix displayed a cytocompatible property, conserved structure, mechanical strength, and mineral content comparable to natural bone. To study new bone formation, implantation of decellularized bone matrix particles seeded with rat mesenchymal stem cells was conducted using an orthotopic in vivo model. After 3 months post-implantation into a critical-sized defect in rat calvaria, new bone was formed around decellularized bone matrix particles and also merged with new bone between decellularized bone matrix particles. New bone formation was analyzed with micro computed tomography, mineral apposition rate, and histomorphometry. Decellularized bone matrix stimulated mesenchymal stem cell proliferation and osteogenic differentiation in vitro and in vivo, achieving effective bone regeneration and thereby serving as a promising biological bone graft.

Hematopoietic stem cells (HSCs) are widely used in transplantation therapy to treat a variety of blood diseases. The success of hematopoietic recovery is of high importance and closely related to the patient's morbidity and mortality after Hematopoietic stem cell transplantation (HSCT). We have previously shown that SALL4 is a potent stimulator for the expansion of human hematopoietic stem/progenitor cells in vitro. In these studies, we demonstrated that systemic administration with TAT-SALL4B resulted in expediting auto-reconstitution and inducing a 30-fold expansion of endogenous HSCs/HPCs in mice exposed to a high dose of irradiation. Most importantly, TAT-SALL4B treatment markedly prevented death in mice receiving lethal irradiation. Our studies also showed that TAT-SALL4B treatment was able to enhance both the short-term and long-term engraftment of human cord blood (CB) cells in NOD/SCID mice and the mechanism was likely related to the in vivo expansion of donor cells in a recipient. This robust expansion was required for the association of SALL4B with DNA methyltransferase complex, an epigenetic regulator critical in maintaining HSC pools and in normal lineage progression. Our results may provide a useful strategy to enhance hematopoietic recovery and reconstitution in cord blood transplantation with a recombinant TAT-SALL4B fusion protein.

Matrix production by nucleus pulposus (NP) cells, the cells residing in the center of the intervertebral disc, can be stimulated by growth factors. Bone morphogenetic proteins (BMPs) hold great promise. Although BMP2 and BMP7 have been used most frequently, other BMPs have also shown potential for NP regeneration. Heterodimers may be more potent than single homodimers, but it is not known whether combinations of homodimers would perform equally well. In this study, we compared BMP2, BMP4, BMP6, and BMP7, their combinations and heterodimers, for regeneration by human NP cells. The BMPs investigated induced variable matrix deposition by NP cells. BMP4 was the most potent, both in the final neotissue glysosaminoglycan content and incorporation efficiency. Heterodimers BMP2/6H and BMP2/7H were more potent than their respective homodimer combinations, but not the BMP4/7H heterodimer. The current results indicate that BMP4 might have a high potential for regeneration of the intervertebral disc. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied.

Current strategies for regeneration of large bone fractures yield limited clinical success mainly due to poor integration and healing. Multidisciplinary approaches in design and development of functional tissue engineered scaffolds are required to overcome these translational challenges. Here, a new generation of hyperelastic bone (HB) implants, loaded with superparamagnetic iron oxide nanoparticles (SPIONs), are 3D bioprinted and their regenerative effect on large non-healing bone fractures is studied. Scaffolds are bioprinted with the geometry that closely correspond to that of the bone defect, using an osteoconductive, highly elastic, surgically friendly bioink mainly composed of hydroxyapatite. Incorporation of SPIONs into HB bioink results in enhanced bacteriostatic properties of bone grafts while exhibiting no cytotoxicity. In vitro culture of mouse embryonic cells and human osteoblast-like cells remain viable and functional up to 14 days on printed HB scaffolds. Implantation of damage-specific bioprinted constructs into a rat model of femoral bone defect demonstrates significant regenerative effect over the 2-week time course. While no infection, immune rejection, or fibrotic encapsulation is observed, HB grafts show rapid integration with host tissue, ossification, and growth of new bone. These results suggest a great translational potential for 3D bioprinted HB scaffolds, laden with functional nanoparticles, for hard tissue engineering applications.

In recent years, multicomponent hydrogels such as interpenetrating polymer networks (IPNs) have emerged as innovative biomaterials due to the synergistic combination of the properties of each network. We hypothesized that an innovative non-animal IPN hydrogel combining self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) with photochemically cross-linkable dextran methacrylate (DexMA) could be a valid alternative to porcine collagen membranes in guided bone regeneration. Calvaria critical-size defects in rabbits were filled with synthetic biphasic calcium phosphate granules in conjunction with Si-HPMC; DexMA; or Si-HPMC/DexMA experimental membranes; and in a control group with a porcine collagen membrane. The synergistic effect obtained by interpenetration of the two polymer networks improved the physicochemical properties, and the gel point under visible light was reached instantaneously. Neutral red staining of murine L929 fibroblasts confirmed the cytocompatibility of the IPN. At 8 weeks, the photo-crosslinked membranes induced a similar degree of mineral deposition in the calvaria defects compared to the positive control, with 30.5 ± 5.2% for the IPN and 34.3 ± 8.2% for the collagen membrane. The barrier effect appeared to be similar in the IPN test group compared with the collagen membrane. In conclusion, this novel, easy-to-handle and apply, photochemically cross-linkable IPN hydrogel is an excellent non-animal alternative to porcine collagen membrane in guided bone regeneration procedures.

Oxygen is critical for optimal bone regeneration. While axolotls and salamanders have retained the ability to regenerate whole limbs, mammalian regeneration is restricted to the distal tip of the digit (P3) in mice, primates, and humans. Our previous study revealed the oxygen microenvironment during regeneration is dynamic and temporally influential in building and degrading bone. Given that regeneration is dependent on a dynamic and changing oxygen environment, a better understanding of the effects of oxygen during wounding, scarring, and regeneration, and better ways to artificially generate both hypoxic and oxygen replete microenvironments are essential to promote regeneration beyond wounding or scarring. To explore the influence of increased oxygen on digit regeneration in vivo daily treatments of hyperbaric oxygen were administered to mice during all phases of the entire regenerative process. Micro-Computed Tomography (μCT) and histological analysis showed that the daily application of hyperbaric oxygen elicited the same enhanced bone degradation response as two individual pulses of oxygen applied during the blastema phase. We expand past these findings to show histologically that the continuous application of hyperbaric oxygen during digit regeneration results in delayed blastema formation at a much more proximal location after amputation, and the deposition of better organized collagen fibers during bone formation. The application of sustained hyperbaric oxygen also delays wound closure and enhances bone degradation after digit amputation. Thus, hyperbaric oxygen shows the potential for positive influential control on the various phases of an epimorphic regenerative response.