ICTV Virus Taxonomy Profile: Poxviridae 2023.

Poxviridae is a family of enveloped, brick-shaped or ovoid viruses. The genome is a linear molecule of dsDNA (128-375 kbp) with covalently closed ends. The family includes the sub-families Entomopoxvirinae, whose members have been found in four orders of insects, and Chordopoxvirinae, whose members are found in mammals, birds, reptiles and fish. Poxviruses are important pathogens in various animals, including humans, and typically result in the formation of lesions, skin nodules, or disseminated rash. Infections can be fatal. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Poxviridae, which is available at ictv.global/report/poxviridae.

Macrocystis pyrifera Lipids Reduce Cytokine-Induced Pro-Inflammatory Signalling and Barrier Dysfunction in Human Keratinocyte Models.

Atopic dermatitis is a chronic condition where epidermal barrier dysfunction and cytokine production by infiltrating immune cells exacerbate skin inflammation and damage. A total lipid extract from Macrocystis pyrifera, a brown seaweed, was previously reported to suppress inflammatory responses in monocytes. Here, treatment of human HaCaT keratinocytes with M. pyrifera lipids inhibited tumour necrosis factor (TNF)-α induced TNF receptor-associated factor 2 and monocyte chemoattractant protein (MCP)-1 protein production. HaCaT cells stimulated with TNF-α, interleukin (IL)-4, and IL-13 showed loss of claudin-1 tight junctions, but little improvement was observed following lipid pre-treatment. Three-dimensional cultures of HaCaT cells differentiated at the air-liquid interface showed increased MCP-1 production, loss of claudin-1 tight junctions, and trans-epidermal leakage with TNF-α, IL-4, and IL-13 stimulation, with all parameters reduced by lipid pre-treatment. These findings suggest that M. pyrifera lipids have anti-inflammatory and barrier-protective effects on keratinocytes, which may be beneficial for the treatment of atopic dermatitis or other skin conditions.

Skin antigen-presenting cells and wound healing: New knowledge gained and challenges encountered using mouse depletion models.

The role of antigen-presenting cells in the skin immune system, in particular Langerhans cells and dendritic cells, has not been well defined. We recently published a study in 'Immunology' where we reported that the loss of langerin-positive cells in the skin accelerated wound repair in the Lang-DTR mouse. The study published here by Li, et al. reports delayed wound closure following depletion of CD11c-positive cells in the CD11c-DTR mouse. In this commentary, we attribute the differences between these results to several factors that differ between the studies including the depletion of different cell populations; differences in the age and the sex of mice; differences in antibiotic use between the studies; and differences in the location of the biopsies that were taken. Here, we describe the impact of these differences on wound healing and conclude that further standardization of the wound model, and further characterization of the specific cells that are depleted in these mice, is necessary to better understand how antigen-presenting cells contribute to wound healing.

Oestrogen deprivation induces chemokine production and immune cell recruitment in in vitro and in vivo models of oestrogen receptor-positive breast cancer.

BACKGROUND: Oestrogen receptor-positive (ER+) breast cancer is commonly treated using endocrine therapies such as aromatase inhibitors which block synthesis of oestradiol, but the influence of this therapy on the immune composition of breast tumours has not been fully explored. Previous findings suggest that tumour infiltrating lymphocytes and immune-related gene expression may be altered by treatment with aromatase inhibitors. However, whether these changes are a direct result of impacts on the host immune system or mediated through tumour cells is not known. We aimed to investigate the effect of oestrogen deprivation on the expression of chemokines and immune infiltration in vitro and in an ER+ immunocompetent mouse model. METHODS: RT-qPCR and a bead-based Bioplex system were used to investigate the expression of chemokines in MCF-7 breast cancer cells deprived of oestrogen. A migration assay and flow cytometry were used to measure the migration of human peripheral blood mononuclear cells (PBMCs) to MCF-7 cells grown without the main biologically active oestrogen, oestradiol. Using flow cytometry and immunohistochemistry, we examined the immune cell infiltrate into tumours created by injecting SSM3 ER+ breast cancer cells into wild-type, immunocompetent 129/SvEv mice. RESULTS: This study demonstrates that oestrogen deprivation increases breast cancer secretion of TNF, CCL5, IL-6, IL-8, and CCL22 and alters total human peripheral blood mononuclear cell migration in an in vitro assay. Oestrogen deprivation of breast cancer cells increases migration of CD4+ T cells and decreases migration of CD11c+ and CD14+ PBMC towards cancer cells. PBMC migration towards breast cancer cells can be reduced by treatment with the non-steroidal anti-inflammatory drugs, aspirin and celecoxib. Treatment with endocrine therapy using the aromatase inhibitor letrozole increases CD4+ T cell infiltration into ER+ breast cancer tumours in immune competent mice. CONCLUSIONS: These results suggest that anti-oestrogen treatment of ER+ breast cancer cells can alter cytokine production and immune cells in the area surrounding the cancer cells. These findings may have implications for the combination and timing of anti-oestrogen therapies with other therapies.

Depletion of langerin+ cells enhances cutaneous wound healing.

Langerin is a C-type lectin receptor that is expressed on Langerhans cells and langerin-positive dermal dendritic cells in the skin. Little is known about the function of langerin+ cells in wound healing. In this study, the effects of ablation of langerin+ cells on healing of a full-thickness excision wound were investigated using the langerin-DTR depletable mouse. Strikingly, depletion of langerin+ cells resulted in more rapid reduction in wound area. Accelerated wound healing in the langerin+ -cell-depleted group was characterized by enhanced neo-epidermis and granulation tissue formation, and increased cellular proliferation within the newly formed tissues. Accelerated healing in the absence of langerin+ cells was associated with increased levels of granulocyte-macrophage colony-stimulating factor, F4/80+ cells and blood vessels within the granulation tissue. These data support an inhibitory role for langerin+ cells during wound healing. Therapies that suppress langerin+ cells or their function may therefore have utility in progressing the healing of wounds in humans.

The role of Langerhans cells in pathologies of the skin.

Langerhans cells (LCs) are epidermal immune cells of myeloid origin. Although these cells were primarily thought to play a defensive role in the skin, evidence now indicates a diverse range of LC-mediated effects including the relay of viral antigens in herpes simplex infection, recruitment of eosinophils in atopic dermatitis and promotion of a Th17 response in Candida infection. LCs may have a protective or suppressive function in pathologies of the skin, with differing functions being driven by the skin milieu. Understanding LC function will help guide the development of interventions that modulate these cells for therapeutic benefit.

The Cutaneous Inflammatory Response to Thermal Burn Injury in a Murine Model.

Many burn interventions aim to target the inflammatory response as a means of enhancing healing or limiting hypertrophic scarring. Murine models of human burns have been developed, but the inflammatory response to injury in these models has not been well defined. The aim of this study was to profile inflammatory cell populations and gene expression relative to healing and scarring in a murine model of thermal burns. Cutaneous injuries were created on the dorsal region of C57Bl/6 mice using a heated metal rod. Animals were euthanized at selected time points over ten weeks, with the lesions evaluated using macroscopic measurements, histology, immunofluorescent histochemistry and quantitative PCR. The burn method generated a reproducible, partial-thickness injury that healed within two weeks through both contraction and re-epithelialization, in a manner similar to human burns. The injury caused an immediate increase in pro-inflammatory cytokine and chemokine expression, coinciding with an influx of neutrophils, and the disappearance of Langerhans cells and mast cells. This preceded an influx of dendritic cells and macrophages, a quarter of which displayed an inflammatory (M1) phenotype, with both populations peaking at closure. As with human burns, the residual scar increased in size, epidermal and dermal thickness, and mast cell numbers over 10 weeks, but abnormal collagen I-collagen III ratios, fibre organization and macrophage populations resolved 3⁻4 weeks after closure. Characterisation of the inflammatory response in this promising murine burn model will assist future studies of burn complications and aid in the preclinical testing of new anti-inflammatory and anti-scarring therapies.

Exploitation of receptor tyrosine kinases by viral-encoded growth factors.

Receptor tyrosine kinases (RTKs) are essential components of cell communication pathways utilized from the embryonic to adult stages of life. These transmembrane receptors bind polypeptide ligands, such as growth factors, inducing signalling cascades that control cellular processes such as proliferation, survival, differentiation, motility and inflammation. Many viruses have acquired homologs of growth factors encoded by the hosts that they infect. Production of growth factors during infection allows viruses to exploit RTKs for entry and replication in cells, as well as for host and environmental dissemination. This review describes the genetic diversity amongst virus-derived growth factors and the mechanisms by which RTK exploitation enhances virus survival, then highlights how viral ligands can be used to further understanding of RTK signalling and function during embryogenesis, homeostasis and disease scenarios.

Short-term treatment of equine wounds with orf virus IL-10 and VEGF-E dampens inflammation and promotes repair processes without accelerating closure.

Healing is delayed in limb wounds relative to body wounds of horses, partly because of sustained inflammation and inefficient angiogenesis. In laboratory animals, proteins derived from orf virus modulate these processes and enhance healing. We aimed to compare immune cell trafficking and the inflammatory, vascular, and epidermal responses in body and limb wounds of horses and then to investigate the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on these processes. Standardized excisional wounds were created on the body and forelimb of horses and their progression monitored macroscopically until healed. Tissue samples were harvested to measure the expression of genes regulating inflammation and repair (quantitative polymerase chain reaction) and to observe epithelialization (histology), innate immune cell infiltration, and angiogenesis (immunofluorescence). Delayed healing of limb wounds was characterized by intensified and extended pro-inflammatory signaling and exacerbated innate immune response, concomitant with the absence of anti-inflammatory eIL-10. Blood vessels were initially more permeable and then matured belatedly, concomitant with retarded production of angiogenic factors. Epithelial coverage was achieved belatedly in limb wounds. Viral proteins were administered to wounds of one body and one limb site/horse at days 1-3, while wounds at matching sites served as controls. Treatment dampened pro-inflammatory gene expression and the innate immune response in all wounds. It also improved angiogenic gene expression, but primarily in body wounds, where it altered blood vessel density and myofibroblast persistence. Moreover, the viral proteins increased epithelialization of all wounds. The short-term viral protein therapy did not, however, improve the healing rate of wounds in either location, likely due to suboptimal dosing. In conclusion, we have further detailed the processes contributing to protracted healing in limb wounds of horses and shown that short-term administration of viral proteins exerts several promising though transient effects that, if optimized, may positively influence healing.

Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

BACKGROUND: Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. HYPOTHESIS/OBJECTIVES: The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. ANIMALS: Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. METHODS: Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. RESULTS: Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. CONCLUSIONS AND CLINICAL IMPORTANCE: This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses.

Effect of a broad-specificity chemokine-binding protein on brain leukocyte infiltration and infarct development.

BACKGROUND AND PURPOSE: Expression of numerous chemokine-related genes is increased in the brain after ischemic stroke. Here, we tested whether post-stroke administration of a chemokine-binding protein (CBP), derived from the parapoxvirus bovine papular stomatitis virus, might reduce infiltration of leukocytes into the brain and consequently limit infarct development. METHODS: The binding spectrum of the CBP was evaluated in chemokine ELISAs, and binding affinity was determined using surface plasmon resonance. Focal stroke was induced in C57Bl/6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion for 23 or 47 hours. Mice were treated intravenously with either bovine serum albumin (10 µg) or CBP (10 µg) at the commencement of reperfusion. At 24 or 48 hours, we assessed plasma levels of the chemokines CCL2/MCP-1 and CXCL2/MIP-2, as well as neurological deficit, brain leukocyte infiltration, and infarct volume. RESULTS: The CBP interacted with a broad spectrum of CC, CXC, and XC chemokines and bound CCL2/MCP-1 and CXCL2/MIP-2 with high affinity (pM range). Stroke markedly increased plasma levels of CCL2/MCP-1 and CXCL2/MIP-2, as well as numbers of microglia and infiltrating leukocytes in the brain. Increases in plasma chemokines were blocked in mice treated with CBP, in which there was reduced neurological deficit, fewer brain-infiltrating leukocytes, and ≈50% smaller infarcts at 24 hours compared with bovine serum albumin-treated mice. However, CBP treatment was no longer protective at 48 hours. CONCLUSIONS: Post-stroke administration of CBP can reduce plasma chemokine levels in association with temporary atten uation of brain inflammation and infarct volume development.

Orf virus IL-10 accelerates wound healing while limiting inflammation and scarring.

Interleukin (IL)-10 plays a critical role in controlling wound inflammation and scar formation. Orf virus, a zoonotic parapoxvirus, induces proliferative skin lesions that resolve with minimal scarring. Orf virus encodes a range of factors that subvert the host's response to infection, including a homolog of IL-10. This study investigated, using a murine full-thickness wound model, whether purified orf virus IL-10 (ovIL-10) can regulate skin repair and scarring. Repeat injections of ovIL-10 into wounded skin accelerated wound closure. Histological analyses of wound sections revealed that treatment with ovIL-10 accelerated wound reepithelialization, granulation tissue coverage of the wound bed, and improved wound revascularization. In addition, wounds treated with ovIL-10 showed a reduction in macrophage infiltration, myofibroblast differentiation, and wound contraction. Treatment of wounds with ovIL-10 also resulted in a reduction in visible scarring that was consistent with the extent of scar tissue formed. Quantitative polymerase chain reaction analysis confirmed that ovIL-10 reduced the expression of key mediators of inflammation and granulation tissue formation. These findings show that ovIL-10, like mammalian IL-10, limits inflammation and scar tissue formation and reveal a new role for both mammalian and viral IL-10 in mediating tissue repair.

Isolation and characterisation of two epithelial-like cell lines from the gills of Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon) and their use in aquatic toxicology.

In vitro gill models are becoming increasingly important in aquatic toxicology, yet the fish gill invitrome is underrepresented, encompassing approximately 0.1% of extant species. Here, we describe the establishment and characterisation of two gill-derived, epithelial-like cell lines isolated from fish species of significant importance to New Zealand: Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon). Designated CAgill1PFR (Chrysophrys auratus, gill 1, Plant & Food Research) and OTgill1PFR (Oncorhynchus tshawytscha, gill 1, Plant & Food Research), these cell lines have each been passaged greater than each 70 times over several years and are considered spontaneously immortalised. Both cell lines required serum for growth and exhibited differential responses to basal media formulations. CAgill1PFR was sensitive to low temperatures (4 °C) but replicated at high temperatures (30 °C), whereas OTgill1PFR was sensitive to high temperatures but remained viable at low temperatures, mirroring the natural environment of their host species. Immunostaining revealed expression of epithelial cell markers cytokeratin and E-cadherin, alongside positivity for the mesenchymal cell marker, vimentin. CAgill1PFR was more sensitive to the environmental toxin 3,4 dichloroaniline than OTgill1PFR through measurements of metabolic activity, membrane integrity, and lysosomal function. Furthermore, CAgill1PFR produced less CYP1A activity, indicative of ongoing biotransformation processes, in response to beta-naphthoflavone than OTgill1PFR. These cell lines expand the toolbox of resources and emphasise the need for species-specific aquatic toxicology research.

The vascular endothelial growth factor (VEGF)-E encoded by orf virus regulates keratinocyte proliferation and migration and promotes epidermal regeneration.

Vascular endothelial growth factor (VEGF)-A, a key regulator of cutaneous blood vessel formation, appears to have an additional role during wound healing, enhancing re-epithelialization. Orf virus, a zoonotic parapoxvirus, induces proliferative skin lesions that initiate in wounds and are characterized by extensive blood vessel formation, epidermal hyperplasia and rete ridge formation. The vascular changes beneath the lesion are largely due to viral-expressed VEGF-E. This study investigated using mouse skin models whether VEGF-E can induce epidermal changes such as that seen in the viral lesion. Injection of VEGF-E into normal skin increased the number of endothelial cells and blood vessels within the dermis and increased epidermal thickening and keratinocyte number. Injection of VEGF-E into wounded skin, which more closely mimics orf virus lesions, increased neo-epidermal thickness and area, promoted rete ridge formation, and enhanced wound re-epithelialization. Quantitative RT-PCR analysis showed that VEGF-E did not induce expression of epidermal-specific growth factors within the wound, but did increase matrix metalloproteinase (MMP)-2 and MMP-9 expression. In cell-based assays, VEGF-E induced keratinocyte migration and proliferation, responses that were inhibited by a neutralizing antibody against VEGF receptor (VEGFR)-2. These findings demonstrate that VEGF-E, both directly and indirectly, regulates keratinocyte function, thereby promoting epidermal regeneration.

Methods to Assess Chemokine Binding and Anti-chemotactic Activity of Virus Proteins.

Chemokines are key instigators of inflammatory and immune responses. Viruses can suppress these responses by secreting proteins that interfere with chemokine action. These proteins bind to chemokines and block the host's ability to recruit immune cells to sites of infection, thus facilitating virus replication and spread. When produced recombinantly, chemokine binding proteins provide a formidable resource to deploy against human disease. Here, we describe an enzyme-linked immunosorbent inhibition assay and a chemotaxis inhibition assay that are employed to assess the chemokine binding strength and anti-chemotactic activity of viral proteins. These assays are quick and reproducible, and are thus ideal for screening putative or modified chemokine binding proteins as the first step in their development as therapeutics.

Parapoxvirus Interleukin-10 Homologues Vary in Their Receptor Binding, Anti-Inflammatory, and Stimulatory Activities.

Homologues of interleukin (IL)-10, a pleiotropic immunomodulatory cytokine, have been identified in the Parapoxvirus genus. The first identified, Orf virus (ORFV) IL-10, greatly enhanced infection of its host, exhibiting immune modulatory effects equivalent to human IL-10. IL-10-like genes were then identified in Bovine papular stomatitis virus (BPSV), Pseudocowpox virus (PCPV), Red deerpox virus (RDPV) and Grey sealpox virus (GSPV). This study aimed to produce and characterise recombinant parapoxvirus IL-10s, then quantitatively compare their receptor binding and immunomodulatory activities. Recombinant IL-10s were expressed, purified, then characterised using bioinformatic, biochemical and enzymatic analyses. Anti-inflammatory effects were assessed in lipoteichoic acid-activated THP-1 monocytes, and stimulatory effects in MC/9 mast cells. IL-10 receptor (IL-10R)1 binding was detected in a competitive displacement assay. BPSV IL-10 inhibited production of monocyte chemoattractant protein (MCP)-1, IL-8 and IL-1ß, induced mast cell proliferation, and bound IL-10R1 similarly to ORFV IL-10. PCPV IL-10 showed reduced MCP-1 inhibition, mast cell proliferation, and IL-10R1 binding. RDPV IL-10 displayed reduced inhibition of IL-8 and MCP-1 production. GSPV IL-10 showed limited inhibition of IL-1ß production and stimulation of mast cell proliferation. These findings provide valuable insight into IL-10 receptor interactions, and suggest that the parapoxvirus IL-10s play similar pathogenic roles during infection of their hosts.

The chemokine-binding protein encoded by the poxvirus orf virus inhibits recruitment of dendritic cells to sites of skin inflammation and migration to peripheral lymph nodes.

Orf virus (ORFV) is a zoonotic parapoxvirus that induces acute pustular skin lesions in sheep and humans. ORFV can reinfect its host and the discovery of several secreted immune modulatory factors that include a chemokine-binding protein (CBP) may explain this phenomenon. Dendritic cells (DC) are professional antigen presenting cells that induce adaptive immunity and their recruitment to sites of infection in skin and migration to peripheral lymph nodes is critically dependent on inflammatory and constitutive chemokine gradients respectively. Here we examined whether ORFV-CBP could disable these gradients using mouse models. Previously we established that ORFV-CBP bound murine inflammatory chemokines with high affinity and here we show that this binding spectrum extends to constitutive chemokines CCL19 and CCL21. Using cell-based chemotaxis assays, ORFV-CBP inhibited the movement of both immature and mature DC in response to these inflammatory and constitutive chemokines respectively. Moreover in C57BL/6 mice, intradermally injected CBP potently inhibited the recruitment of blood-derived DC to lipopolysaccharide-induced sites of skin inflammation and inhibited the migration of ex vivo CpG-activated DC to inguinal lymph nodes. Finally we showed that ORFV-CBP completely inhibited T responsiveness in the inguinal lymph nodes using intradermally injected DC pulsed with ovalbumin peptide and transfused transgenic T cells.

Advancements in the Delivery of Growth Factors and Cytokines for the Treatment of Cutaneous Wound Indications.

Significance: Wound healing involves the phasic production of growth factors (GFs) and cytokines to progress an acute wound to a resolved scar. Dysregulation of these proteins contributes to both wound chronicity and excessive scarring. Direct supplementation of GFs and cytokines for treatment of healing and scarring complications has, however, been disappointing. Failings likely relate to an inability to deliver recombinant proteins at physiologically relevant levels to an environment conducive to healing. Recent Advances: Inspired by the extracellular matrix, natural biomaterials have been developed that resemble human skin, and are capable of delivering bioactives. Hybrid biomaterials made using multiple polymers, fabrication methods, and proteins are proving efficacious in animal models of acute and impaired wound healing. Critical Issues: For clinical translation, these delivery systems must be tailored for specific wound indications and the correct phase of healing. GFs and cytokines must be delivered in a controlled manner that will target specific healing or scarring impairments. Preclinical assessment in clinically relevant animal models of impaired or excessive healing is critical. Future Directions: Clinical success will likely depend on the GF or cytokine selected, their compatibility with the chosen biomaterial(s), degradation rate of the fabricated system, and the degree of control over release kinetics. Further testing is essential to assess which wound indications are most suited to specific delivery systems and to prove whether they provide superior efficacy over direct protein therapies.

Murine Model of Thermal Burn Injury for Evaluating Protein Therapeutics Derived from Viruses.

In vivo wound healing models are predictive preclinical tests for therapeutics that enhance skin repair or limit scarring. Large animals, such as swine, heal in a manner similar to humans, but testing is impractical and expensive. Experiments in mice are more economic, but may be less translatable as this species heals primarily through contraction, not by the processes of epithelialization and granulation tissue formation as seen in human wounds. Here, we describe a murine model of thermal burn injury that closely mimics human healing, resulting in a large, hypertrophic-like scar. This practical, reproducible model is ideal for testing promising wound-healing therapies, such as virus-derived growth factors and immune-modulatory proteins.

Strategies for inclusion of growth factors into 3D printed bone grafts.

There remains a critical need to develop new technologies and materials that can meet the demands of treating large bone defects. The advancement of 3-dimensional (3D) printing technologies has allowed the creation of personalized and customized bone grafts, with specific control in both macro- and micro-architecture, and desired mechanical properties. Nevertheless, the biomaterials used for the production of these bone grafts often possess poor biological properties. The incorporation of growth factors (GFs), which are the natural orchestrators of the physiological healing process, into 3D printed bone grafts, represents a promising strategy to achieve the bioactivity required to enhance bone regeneration. In this review, the possible strategies used to incorporate GFs to 3D printed constructs are presented with a specific focus on bone regeneration. In particular, the strengths and limitations of different methods, such as physical and chemical cross-linking, which are currently used to incorporate GFs to the engineered constructs are critically reviewed. Different strategies used to present one or more GFs to achieve simultaneous angiogenesis and vasculogenesis for enhanced bone regeneration are also covered in this review. In addition, the possibility of combining several manufacturing approaches to fabricate hybrid constructs, which better mimic the complexity of biological niches, is presented. Finally, the clinical relevance of these approaches and the future steps that should be taken are discussed.