Segmental Additive Tissue Engineering

Bioreactor systems, for example, spinner flask and perfusion bioreactors, and cell-seeded three-dimensional (3D)-printed scaffolds are used in bone tissue engineering strategies to stimulate cells and produce bone tissue suitable for implantation into the patient. The construction of functional and clinically relevant bone graft using cell-seeded 3D-printed scaffolds within bioreactor systems is still a challenge. Bioreactor parameters, for example, fluid shear stress and nutrient transport, will crucially affect cell function on 3D-printed scaffolds. Therefore, fluid shear stress induced by spinner flask and perfusion bioreactors might differentially affect osteogenic responsiveness of pre-osteoblasts inside 3D-printed scaffolds. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine fluid shear stress and osteogenic responsiveness of MC3T3-E1 pre-osteoblasts seeded on the scaffolds in the bioreactors using finite element (FE)-modeling and experiments. FE-modeling was used to quantify wall shear stress (WSS) distribution and magnitude inside 3D-printed PCL scaffolds within spinner flask and perfusion bioreactors. MC3T3-E1 pre-osteoblasts were seeded on NaOH surface-modified 3D-printed PCL scaffolds, and cultured in customized static, spinner flask, and perfusion bioreactors up to 7 days. The scaffolds' physicochemical properties and pre-osteoblast function were assessed experimentally. FE-modeling showed that spinner flask and perfusion bioreactors locally affected WSS distribution and magnitude inside the scaffolds. The WSS distribution was more homogeneous inside scaffolds in perfusion than in spinner flask bioreactors. The average WSS on scaffold-strand surfaces ranged from 0 to 6.5 mPa for spinner flask bioreactors, and from 0 to 4.1 mPa for perfusion bioreactors. Surface modification of scaffolds by NaOH resulted in a surface with a honeycomb-like pattern and increased surface roughness (1.6-fold), but decreased water contact angle (0.3-fold). Both spinner flask and perfusion bioreactors increased cell spreading, proliferation, and distribution throughout the scaffolds. Perfusion, but not spinner flask bioreactors more strongly enhanced collagen (2.2-fold) and calcium deposition (2.1-fold) throughout the scaffolds after 7 days compared with static bioreactors, likely due to uniform WSS-induced mechanical stimulation of the cells revealed by FE-modeling. In conclusion, our findings indicate the importance of using accurate FE models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems. Impact Statement The success of cell-seeded three-dimensional (3D)-printed scaffolds depends on cell stimulation by biomechanical/biochemical factors to produce bone tissue suitable for implantation into the patient. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine wall shear stress (WSS) and osteogenic responsiveness of pre-osteoblasts seeded on the scaffolds using finite element (FE)-modeling and experiments. We found that cell-seeded 3D-printed PCL scaffolds within perfusion bioreactors more strongly enhanced osteogenic activity than within spinner flask bioreactors. Our results indicate the importance of using accurate FE-models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems.

Tissue engineering (by culturing cells on appropriate scaffolds, and using bioreactors to drive the correct bone structure formation) is an attractive alternative to bone grafting or implantation of bone substitutes. Osteogenesis is a biological process that involves many molecular intracellular pathways organized to optimize bone modeling. The use of bioreactor systems and especially the perfusion bioreactor, provides both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In this mini-review all the characteristics for the production of an appropriate bone construct are analyzed: the stem cell source, scaffolds useful for the seeding of pre-osteoblastic cells and the effects of fluid flow on differentiation and proliferation of bone precursor cells. By automating and standardizing tissue manufacture in controlled closed systems, engineered tissues may reduce the gap between the process of bone formation in vitro and subsequent graft of bone substitutes in vivo. Keywords: Biomaterials, extracellular matrix, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSC), osteogenesis, perfusion bioreactor, scaffolds, tissue engineering

Several conditions in clinical orthopaedic practice can lead to the development of a diaphyseal segmental bone defect, which cannot heal without intervention. Segmental bone defects have been traditionally treated with bone grafting and/or distraction osteogenesis, methods that have many advantages, but also major drawbacks, such as limited availability, risk of disease transmission and prolonged treatment. In order to overcome such limitations, biological treatments have been developed based on specific pathways of bone physiology and healing. Bone tissue engineering is a dynamic field of research, combining osteogenic cells, osteoinductive factors, such as bone morphogenetic proteins, and scaffolds with osteoconductive and osteoinductive attributes, to produce constructs that could be used as bone graft substitutes for the treatment of segmental bone defects. Scaffolds are usually made of ceramic or polymeric biomaterials, or combinations of both in composite materials. The purpose of the present review is to discuss in detail the molecular and cellular basis for the development of bone tissue engineering constructs.

The management of segmental bone defects presents a complex reconstruction challenge for orthopedic surgeons. Current treatment options are limited by efficacy across the spectrum of injury, morbidity, and cost. Regional gene therapy is a promising tissue engineering strategy for bone repair, as it allows for local implantation of nucleic acids or genetically modified cells to direct specific protein expression. In cell-based gene therapy approaches, a variety of different cell types have been described including mesenchymal stem cells (MSCs) derived from multiple sources-bone marrow, adipose, skeletal muscle, and umbilical cord tissue, among others. MSCs, in particular, have been well studied, as they serve as a source of osteoprogenitor cells in addition to providing a vehicle for transgene delivery. Furthermore, MSCs possess immunomodulatory properties, which may support the development of an allogeneic "off-the-shelf" gene therapy product. Identifying an optimal cell type is paramount to the successful clinical translation of cell-based gene therapy approaches. Here, we review current strategies for the management of segmental bone loss in orthopedic surgery, including bone grafting, bone graft substitutes, and operative techniques. We also highlight regional gene therapy as a tissue engineering strategy for bone repair, with a focus on cell types and cell sources suitable for this application.

Tissue and cell engineering.

Strategies to optimize engineered tissue towards native human aortic valves

High-Speed-Microscopy for Scalable Quality Control in Automated Production of Stem Cell Spheroids for Tissue Engineering

Mesenchymal stem cell-based nanoparticles and scaffolds in regenerative medicine

Reconstructions of segmental diaphyseal bone defects with massive allografts are related to complications like nonunion and fractures. A reconstruction of these defects with a cage filled with an impacted morsellized bone graft could be an alternative. The bone graft in these cages should ideally be loaded to prevent resorption. Loading of morsellized bone grafts however can cause instability. The goal of this study was to assess the stability of an impacted morsellized bone graft in a cage under dynamic loaded conditions in an in vitro reconstruction of a segmental diaphyseal bone defect. The second goal was to assess the influence of cage type, washing of the graft and bone-cage fit. Two different cage types were filled with impacted morsellized bone graft. The grafts were used washed and unwashed and in variable bone-cage fit conditions. We recorded the bone graft deformation in the cage under dynamic loaded conditions. Stability appeared to be not very sensitive to the cage type and whether the bone chips were washed or not. However, quality of the fit of the cage with the bone segment was an important parameter and should be optimized during surgery. Morsellized impacted bone graft in a cage is stable in dynamic loaded conditions in an in vitro reconstruction of a segmental diaphyseal bone defect. We believe that this method of reconstruction is a promising alternative for the reconstruction of large diaphyseal bone defects and should be tested relative to its biological merits in animal experiments.

This study evaluated whether polymicrobial infection affects reoperation rates due to infection recurrence and treatment failure with the Masquelet technique in infected posttraumatic segmental bone defects of the femur and tibia. We retrospectively analyzed patients treated between 2012 and 2021 in two trauma referral centers. We evaluated demographic data, injury, treatment, infection recurrence, failures, and bone healing rates according to whether the infection was mono- or polymicrobial. After uni-bivariate analysis between patients with polymicrobial and monomicrobial infection, we identified the variables associated with infection recurrence and failure through multivariate analysis. We analyzed 54 patients, 30 (55.55%) with tibial and 24 (44.44%) femoral segmental bone defects, with a mean follow-up of 41.7 ± 15.0months. Forty-four (81.48%) presented monomicrobial, and 10 (18.51%) polymicrobial infections. Comparatively, the need for soft tissue reconstruction and the infection recurrence rate was significantly higher in patients with polymicrobial infections. There was no significant difference in the failure rate (20 vs. 6.81% p = 0.23). Multivariable logistic regression analysis identified the polymicrobial infection as the only independent variable associated with infection recurrence (Odds Ratio = 11.07; p = 0.0017). Our analysis suggests that polymicrobial infection is associated with a higher risk of infection recurrence in treating the femur and tibia segmental bone defects with the Masquelet technique. This information can help surgeons to inform patients about this and give them a realistic expectation of the outcome and the possibility of reoperation.

Endothelial progenitor cells (EPCs) can be defined as bone marrow-derived precursor cells with the ability to differentiate into endothelial cells and to participate in the formation of new blood vessels. Bone marrow is the primary source of endothelial progenitors, which can be mobilized to the peripheral circulation and may seed in remote organs and tissues such as liver, spleen, heart, muscle, and adipose tissue. EPCs have been shown to express various endothelial surface markers, such as CD34, VEGFR2, and CD133, and to home to sites of ischemia1 (Figure 1).Figure 1: Phenotype of cultured endothelial progenitor cells (EPCs) can be characterized according to their ability to uptake Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) to confirm their endothelial lineage. Image demonstrates Dil-Ac-LDL-stained EPCs under fluorescent microscope (×40).Vascular ingrowth at the fracture site has a cardinal role in the healing process and regeneration of the bone following fracture. However, segmental bone defects after severe trauma, infection, and surgical removal of tumors remain a major clinical problem. These facts, together with reports documenting the remarkable therapeutic potential of EPCs to improve neovascularization and tissue perfusion in other disciplines, have been the major drivers recently to investigate the effects of EPC therapy on fracture healing. Matsumoto et al2 investigated the therapeutic potential of systemically administered CD34+ cells on fracture healing in a rodent model. The authors transplanted human peripheral blood CD34+ cells, mononuclear cells, or saline into immunodeficient rats with a non-healing femoral fracture. Fracture healing was significantly enhanced in the CD34+ group compared with the mononuclear cell and saline groups. Laser Doppler imaging demonstrated that fracture-induced ischemia was significantly reduced in the CD34+ cell-transplanted group compared with the other groups. The authors also noted that approximately 20% of human peripheral blood CD34+ cells expressed mRNA for osteocalcin after transplantation to a fracture site; this outcome may indicate the potential of CD34+ cells for osteogenic and endothelial differentiation. Atesok et al3 evaluated the effects of the local use of ex vivo-expanded EPCs on the stimulation of angiogenesis and the promotion of bone healing at a fracture site in a rat femur osteotomy model. Based on the results of radiographic, histologic, and micro-CT comparisons of the EPC-treated group with a control group, the authors stated that “local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.” In a similar study, the same research group reported that local EPC therapy favorably affects biomechanical stability.4 Recent reports also suggest that EPCs derived from peripheral blood contribute to osteogenic differentiation by mesenchymal stem cells (MSCs) in vitro and that MSCs support the proliferation of EPCs.5 Aguirre et al6 investigated the interactions between bone marrow EPCs (BM-EPCs) and MSCs in an in vitro co-culture system. Their data suggested that cross-talk occurs between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms, leading to modulation of the angiogenic response. In a rat model study, Seebach et al7 observed a synergistic effect between EPCs and MSCs and suggested that the initial stage of neovascularization by EPCs be considered crucial for complete bone regeneration. Positive effects of EPC therapy have been demonstrated in ligament tissue regeneration, as well. Matsumoto et al8 recently demonstrated that CD34- and CD146-expressing vascular cells exist in human anterior cruciate ligament tissues, have a potential for multilineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured anterior cruciate ligament. In a rodent model, Tei et al9 studied the effects of locally transplanted human peripheral blood CD34+ cells on the healing of medial collateral ligament injury. Macroscopic, histologic, and biomechanical assessments showed significantly enhanced ligament healing in a CD34+ cell transplantation group compared with a control group. The authors suggested that “local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.” Based on the promising results from ex vivo and animal model EPC studies, clinical trials have been started. As a pilot case from a clinical trial, Kuroda et al10 reported the results of transplantation of autologous peripheral blood CD34+ cells—the hematopoietic/EPC-enriched population—into a patient with nonunion of a tibia fracture. Clinical and radiologic healing of the fracture was achieved at 12 weeks after the cell therapy with bone grafting, and no serious short-term complications were encountered. EPCs, with their unique features, such as the ability to differentiate into endothelial cells and to participate in the establishment of neovasculature, in addition to their high plasticity, may offer therapeutic alternatives for both bone and ligament tissue engineering. It is likely that more investigators will be attracted in the near future to exploring the potential of these cells in orthopaedic surgery.11

Maximizing cartilage formation and integration via a trajectory-based tissue engineering approach

Segmental bone defects represent a complex clinical challenge in reconstructive orthopedics, etiologically associated with high-energy trauma, oncological resections, and osteomyelitic processes. The two-stage Masquelet technique with induced membrane formation demonstrates high efficacy in reconstructing critical-size bone defects, however, the molecular biochemical mechanisms of reparative osteogenesis during its application remain insufficiently investigated. Objective. To evaluate the dynamics of biochemical markers of bone metabolism in patients with segmental defects of long tubular bones during treatment using the induced membrane (Masquelet) technique in order to determine the metabolic characteristics of osteogenesis and to optimize bone regeneration. Methods. The study included 85 patients aged 18 to 65 years. The experimental group consisted of 44 patients with segmental bone defects, while the control group comprised 41 patients with low-energy fractures. Biochemical markers such as alkaline phosphatase, osteocalcin, β-CrossLaps, calcium, phosphorus, and parathyroid hormone were analyzed. Results. Before treatment, significant abnormalities in bone metabolism markers were observed in the experimental group: elevated alkaline phosphatase (175.45 ± 46.2) U/L, osteocalcin (53.70 ± 12.4) ng/mL, β-CrossLaps (0.949 ± 0.271) ng/mL. Following treatment, 75-92 % of patients demonstrated normalization of biochemical parameters, indicating stimulation of reparative osteogenesis. Conclusions. The Masquelet technique effectively stimulates bone regeneration in segmental defects, as confirmed by the normalization of biochemical markers. Comprehensive biochemical monitoring can serve as a valuable tool for assessing treatment efficacy.

Objective To investigate the principles and effects of cancellous bone wrapping grafting for treatment of segmental defects of long bone. Methods From January 2008 to December 2015, 50 patients with segmental long bone defect were treated by various wrapping grafting with rich autogenous can-cellous bone. They were 31 males and 19 females, aged from 13 to 69 years (average, 34.6 years). The cancellous bone was wrapped by titanium mesh in 8 cases, by wire mesh in 10, by line binding in 13, and by induced membrane in 19. The bone defect was located at tibia in 22 cases, at radius in 10, at humerus in 8, at ulna in 7 and at femur in 3. The length of bone defect ranged from 3 to 9 cm, averaging 5.9 cm. Bone healing, complications and functionary recovery of adjacent joint were recorded. The bone defect healing and functionary recovery of adjacent joint were evaluated according to the Paley criteria. Results The inci-sions healed by the first intention in 48 cases and by the second in 2. All were followed up for 12 to 48 months (average, 19.1 months). All the bone defects healed by the first intention but one treated by induced membrane wrapping which was healed 15 months later by the secondary grafting due to nonunion at ends. The total clinical healing time ranged from 3 to 16 months (average, 6.1 months). The last follow-ups showed that all the affected limbs resumed weight-bearing activities. The healing of bone defects was graded as excellent in all but one in the group of induced membrane wrapping. Totally, the functionary recovery of adjacent joint was excellent in 18, good in 22, fair in 7 and poor in 3 cases (an excellent and good rate of 80.0%). Conclusions Cancellous bone wrapping grafting can avoid or significantly reduce loosening and absorption of cancellous bone graft after tradi-tional bone grafting. Although it is effective for treatment of large segmental bone defect, its methods should vary according to the specific conditions of the patient. Key words: Bones; Fractures, ununited; Free bone graft; Wrapping bone graft

Efficacy of total flavonoids of Rhizoma drynariae on the blood vessels and the bone graft in the induced membrane