Impact of NaOH based perfusion-decellularization protocol on mechanical resistance of structural bone allografts.

INTRODUCTION: To mitigate the post-operative complication rates associated with massive bone allografts, tissue engineering techniques have been employed to decellularize entire bones through perfusion with a sequence of solvents. Mechanical assessment was performed in order to compare conventional massive bone allografts and perfusion/decellularized massive bone allografts. MATERIAL AND METHODS: Ten porcine femurs were included. Five were decellularized by perfusion. The remaining 5 were left untreated as the "control" group. Biomechanical testing was conducted on each bone, encompassing five different assessments: screw pull-out, 3-points bending, torsion, compression and Vickers indentation. RESULTS: Under the experimental conditions of this study, all five destructive tested variables (maximum force until screw pull-out, maximum elongation until screw pull-out, energy to pull out the screw, fracture resistance in flexion and maximum constrain of compression) were statistically significantly superior in the control group. All seven nondestructive variables (Young's modulus in flexion, Young's modulus in shear stress, Young's modulus in compression, Elastic conventional limit in compression, lengthening to rupture in compression, resilience in compression and Vickers Hardness) showed no significant difference. DISCUSSION: Descriptive statistical results suggest a tendency for the biomechanical characteristics of decellularized bone to decrease compared with the control group. However, statistical inferences demonstrated a slight significant superiority of the control group with destructive mechanical stresses. Nondestructive mechanical tests (within the elastic phase of Young's modulus) were not significantly different.

Collagen molecular organization preservation in human fascia lata and periosteum after tissue engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25 kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (-32.6%) were recorded, as well as less collagen enzymatic crosslinks (-25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (-37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (-29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

Multiple Joint Replacements for Progressive Pseudorheumatoid Dysplasia: A Case Report.

Introduction: Progressive pseudorheumatoid dysplasia is a rare autosomal recessive disorder caused by a mutation in the Wnt1-inducible signaling protein 3 gene, with few cases reported. Case Report: We discuss the case of a 19-year-old Caucasian male patient with polyarticular involvement, including shoulders, elbows, wrists, hands, spine, hips, knees, and notably the ankles. Despite a well-conducted medical treatment, due to the rapid progression of his condition, the patient underwent bilateral total hip and knee arthroplasties, as well as left ankle replacement. Conclusion: This case report highlights the importance of conducting the diagnosis of these rare diseases and the important place of joint replacements in the recovery of joint functions, even in young patients.

Decellularization of Massive Bone Allografts By Perfusion: A New Protocol for Tissue Engineering.

In terms of large bone defect reconstructions, massive bone allografts may sometimes be the only solution. However, they are still burdened with a high postoperative complication rate. Our hypothesis is that the immunogenicity of residual cells in the graft is involved in this issue. Decellularization by perfusion might therefore be the answer to process and create more biologically effective massive bone allografts. Seventy-two porcine bones were used to characterize the efficiency of our sodium hydroxide-based decellularization protocol. A sequence of solvent perfusion through each nutrient artery was set up to ensure the complete decellularization of whole long bones. Qualitative (histology and immunohistochemistry [IHC]) and quantitative (fluoroscopic absorbance and enzyme-linked immunosorbent assay) evaluations were performed to assess the decellularization and the preservation of the extracellular matrix in the bone grafts. Cytotoxicity and compatibility were also tested. Comparatively to nontreated bones, our experiments showed a very high decellularization quality, demonstrating that perfusion is mandatory to achieve an entire decellularization. Moreover, results showed a good preservation of the bone composition and microarchitecture, Haversian systems and vascular network included. This protocol reduces the human leukocyte antigen antigenic load of the graft by >50%. The majority of measured growth factors is still present in the same amount in the decellularized bones compared to the nontreated bones. Histology and IHC show that the bones were cell compatible, noncytotoxic, and capable of inducing osteoblastic differentiation of mesenchymal stem cells. Our decellularization/perfusion protocol allowed to create decellularized long bone graft models, thanks to their inner vascular network, ready for in vivo implantation or to be further used as seeding matrices.

Delayed Spontaneous Fracture of the Proximal Tibia after Tibial Tubercle Osteotomy for Treatment of Patella Instability: A Case Report.

Introduction: Tibial tubercle osteotomy (TTO) is a surgical procedure commonly used. Several local postoperative complications have been identified in the literature, such as early proximal tibial fractures. In exceptional cases, these fractures can occur later in the postoperative period, i.e., after 6 months. The current paper described a case study of this rare delayed complication. Case Report: An athletic 24-year-old military man was admitted to the emergency department following a spontaneous proximal tibial fracture below and arising from the TTO site realized 7 months earlier. Conclusion: Delayed spontaneous fractures of the proximal tibia following TTO are extremely rare complications. This paper describes a case of delayed spontaneous fracture in a young athlete. Factors contributing to this fracture are analyzed. An adaptation of the surgical technique and rehabilitation protocol is discussed.

Adequacy of in-mission training to treat tibial shaft fractures in mars analogue testing.

Long bone fractures are a concern in long-duration exploration missions (LDEM) where crew autonomy will exceed the current Low Earth Orbit paradigm. Current crew selection assumptions require extensive complete training and competency testing prior to flight for off-nominal situations. Analogue astronauts (n = 6) can be quickly trained to address a single fracture pattern and then competently perform the repair procedure. An easy-to-use external fixation (EZExFix) was employed to repair artificial tibial shaft fractures during an inhabited mission at the Mars Desert Research Station (Utah, USA). Bone repair safety zones were respected (23/24), participants achieved 79.2% repair success, and median completion time was 50.04 min. Just-in-time training in-mission was sufficient to become autonomous without pre-mission medical/surgical/mechanical education, regardless of learning conditions (p > 0.05). Similar techniques could be used in LDEM to increase astronauts' autonomy in traumatic injury treatment and lower skill competency requirements used in crew selection.

An Easy-To-Use External Fixator for All Hostile Environments, from Space to War Medicine: Is It Meant for Everyone's Hands?

Long bone fractures in hostile environments pose unique challenges due to limited resources, restricted access to healthcare facilities, and absence of surgical expertise. While external fixation has shown promise, the availability of trained surgeons is limited, and the procedure may frighten unexperienced personnel. Therefore, an easy-to-use external fixator (EZExFix) that can be performed by nonsurgeon individuals could provide timely and life-saving treatment in hostile environments; however, its efficacy and accuracy remain to be demonstrated. This study tested the learning curve and surgical performance of nonsurgeon analog astronauts (n = 6) in managing tibial shaft fractures by the EZExFix during a simulated Mars inhabited mission, at the Mars Desert Research Station (Hanksville, UT, USA). The reduction was achievable in the different 3D axis, although rotational reductions were more challenging. Astronauts reached similar bone-to-bone contact compared to the surgical control, indicating potential for successful fracture healing. The learning curve was not significant within the limited timeframe of the study (N = 4 surgeries lasting <1 h), but the performance was similar to surgical control. The results of this study could have important implications for fracture treatment in challenging or hostile conditions on Earth, such as war or natural disaster zones, developing countries, or settings with limited resources.

How to avoid systematic postoperative blood test after total hip arthroplasty: A new risk scoring system compared to Wu's score.

BACKGROUND: Routine laboratory studies are often performed following total hip arthroplasty (THA). However, lately, their necessity has been challenged and risk factors for postoperative transfusion are still debated. Recently, a risk scoring system to single out patients that should have a postoperative blood test has been published by Wu et al. The purposes of this retrospective study were: (1) to validate this recently published risk scoring system to identify patients who should have a postoperative laboratory test; (2) to single out risk factors of postoperative transfusion; (3) to determine if another score can more accurately predict the need for postoperative transfusion. HYPOTHESIS: Wu et al.'s risk scoring system can accurately identify patients who should have a postoperative blood test. METHODS: In all, 1693 patients who underwent primary THAs between June 2015 and October 2020 were screened for potential eligibility to include 1000 patient for analysis. Preoperative and postoperative blood tests were done for every patient. Clinical information and laboratory results were retrospectively collected and analyzed. A descriptive analysis followed by univariate and multivariate analysis were sequentially performed. A multiple logistic regression model was employed to determine a formula predicting the transfusion risk called THABUS for Total Hip Arthroplasty Blood test Usefulness Score. The risk scoring system for complete blood count published by Wu et al. in may 2020 was performed for every patient and compared to the THABUS predictive model. RESULTS: The transfusion rate was 2.3% (23/1000). The risk-scoring system published by Wu and al. showed that a laboratory test was necessary for 60.6% (606/1000) however 13% (3/23) of the patients who needed a blood transfusion were missed by the risk-scoring system, giving it a sensitivity of 86.95% and a specificity of 40%. Increasing age, arterial hypertension, female gender, low preoperative hemoglobin, ASA score≥2 and diagnosis of osteonecrosis of the femoral head were significantly associated with postoperative transfusion. The THABUS formula can predict the risk for transfusion with a sensibility of 96.65% and a specificity of 75.54%. In our cohort of 1000 patients, following the THABUS formula would have led to 261 postoperative blood test and cost savings of 32,132$. Only one patient (4.3%) was missed by our new score. The THABUS formula is significantly better than Wu et al.'s complete blood count score in identifying both patient that will need a transfusion (p<0.01) and those who shouldn't have a postoperative blood test (p<0.001). Medical intervention because of creatinine or electrolytes abnormality was needed in 0.3% (3/1000) of patients. DISCUSSION: In this study Wu et al.'s recently published complete blood count risk-scoring system was not validated. However, in the studied population the THABUS formula can accurately target patients who might need a transfusion. The use of the THABUS formula could reduce hospitalization costs without compromising the patients' safety. LEVEL OF EVIDENCE: III, case-control study.

A New Osteogenic Membrane to Enhance Bone Healing: At the Crossroads between the Periosteum, the Induced Membrane, and the Diamond Concept.

The lack of viability of massive bone allografts for critical-size bone defect treatment remains a challenge in orthopedic surgery. The literature has reviewed the advantages of a multi-combined treatment with the synergy of an osteoconductive extracellular matrix (ECM), osteogenic stem cells, and growth factors (GFs). Questions are still open about the need for ECM components, the influence of the decellularization process on the latter, the related potential loss of function, and the necessity of using pre-differentiated cells. In order to fill in this gap, a bone allograft surrounded by an osteogenic membrane made of a decellularized collagen matrix from human fascia lata and seeded with periosteal mesenchymal stem cells (PMSCs) was analyzed in terms of de-/recellularization, osteogenic properties, PMSC self-differentiation, and angiogenic potential. While the decellularization processes altered the ECM content differently, the main GF content was decreased in soft tissues but relatively increased in hard bone tissues. The spontaneous osteogenic differentiation was necessarily obtained through contact with a mineralized bone matrix. Trying to deepen the knowledge on the complex matrix-cell interplay could further propel these tissue engineering concepts and lead us to provide the biological elements that allow bone integration in vivo.

Reconstruction of the human nipple-areolar complex: a tissue engineering approach.

Introduction: Nipple-areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization-recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast. Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30 days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7 days, while human keratinocytes were seeded on the scaffold epidermal side for 10 days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis. Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30 days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7 days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7 days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10 days of culture. Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

Periosteum and fascia lata: Are they so different?

Introduction: The human fascia lata (HFL) is used widely in reconstructive surgery in indications other than fracture repair. The goal of this study was to compare microscopic, molecular, and mechanical properties of HFL and periosteum (HP) from a bone tissue engineering perspective. Material and Methods: Cadaveric HP and HFL (N = 4 each) microscopic morphology was characterized using histology and immunohistochemistry (IHC), and the extracellular matrix (ECM) ultrastructure assessed by means of scanning electron microscopy (SEM). DNA, collagen, elastin, glycosaminoglycans, major histocompatibility complex Type 1, and bone morphogenetic protein (BMP) contents were quantified. HP (N = 6) and HFL (N = 11) were submitted to stretch tests. Results: Histology and IHC highlighted similarities (Type I collagen fibers and two-layer organization) but also differences (fiber thickness and compaction and cell type) between both tissues, as confirmed using SEM. The collagen content was statistically higher in HFL than HP (735 vs. 160.2 µg/mg dry weight, respectively, p < 0.0001). On the contrary, DNA content was lower in HFL than HP (404.75 vs. 1,102.2 µg/mg dry weight, respectively, p = 0.0032), as was the immunogenic potential (p = 0.0033). BMP-2 and BMP-7 contents did not differ between both tissues (p = 0.132 and p = 0.699, respectively). HFL supported a significantly higher tension stress than HP. Conclusion: HP and HFL display morphological differences, despite their similar molecular ECM components. The stronger stretching resistance of HFL can specifically be explained by its higher collagen content. However, HFL contains many fewer cells and is less immunogenic than HP, as latter is rich in periosteal stem cells. In conclusion, HFL is likely suitable to replace HP architecture to confer a guide for bone consolidation, with an absence of osteogenicity. This study could pave the way to a bio-engineered periosteum built from HFL.

Decellularized vascularized bone grafts: A preliminary in vitro porcine model for bioengineered transplantable bone shafts.

Introduction: Durable reconstruction of critical size bone defects is still a surgical challenge despite the availability of numerous autologous and substitute bone options. In this paper, we have investigated the possibility of creating a living bone allograft, using the perfusion/decellularization/recellularization (PDR) technique, which was applied to an original model of vascularized porcine bone graft. Materials and Methods: 11 porcine bone forelimbs, including radius and ulna, were harvested along with their vasculature including the interosseous artery and then decellularized using a sequential detergent perfusion protocol. Cellular clearance, vasculature, extracellular matrix (ECM), and preservation of biomechanical properties were evaluated. The cytocompatibility and in vitro osteoinductive potential of acellular extracellular matrix were studied by static seeding of NIH-3T3 cells and porcine adipose mesenchymal stem cells (pAMSC), respectively. Results: The vascularized bone grafts were successfully decellularized, with an excellent preservation of the 3D morphology and ECM microarchitecture. Measurements of DNA and ECM components revealed complete cellular clearance and preservation of ECM's major proteins. Bone mineral density (BMD) acquisitions revealed a slight, yet non-significant, decrease after decellularization, while biomechanical testing was unmodified. Cone beam computed tomography (CBCT) acquisitions after vascular injection of barium sulphate confirmed the preservation of the vascular network throughout the whole graft. The non-toxicity of the scaffold was proven by the very low amount of residual sodium dodecyl sulfate (SDS) in the ECM and confirmed by the high live/dead ratio of fibroblasts seeded on periosteum and bone ECM-grafts after 3, 7, and 16 days of culture. Moreover, cell proliferation tests showed a significant multiplication of seeded cell populations at the same endpoints. Lastly, the differentiation study using pAMSC confirmed the ECM graft's potential to promote osteogenic differentiation. An osteoid-like deposition occurred when pAMSC were cultured on bone ECM in both proliferative and osteogenic differentiation media. Conclusion: Fully decellularized bone grafts can be obtained by perfusion decellularization, thereby preserving ECM architecture and their vascular network, while promoting cell growth and differentiation. These vascularized decellularized bone shaft allografts thus present a true potential for future in vivo reimplantation. Therefore, they may offer new perspectives for repairing large bone defects and for bone tissue engineering.

Treatment of open tibia fractures in Sub-Saharan African countries : a systematic review.

Open tibia fracture (OTF) treatment is well documented in developed countries. Yet, this fracture pattern remains challenging because it is associated with an increased risk of infection and delayed union, particularly in case of Gustilo III B and C open fractures. Since access to healthcare is limited in Sub- Saharan African countries, this paper explores the results of OTF management in this setting. A systematic review of the literature was conducted using current databases such as MEDLINE, Cochrane, EMBASE, PubMed, ScienceDirect, Scopus, and Google Scholar in order to identify prospective studies with cohorts of patients treated for OTF. Studies were included based on predefined inclusion and exclusion criteria. The quality of studies was analyzed by the Coleman Methodology Score (CMS). Eight papers met the inclusion criteria and had an average CMS of 70 (range 54-73). The most common treatment was non-operative management of the fracture with cast immobilization (67%). Gustilo Type II and III fractures were associated with a higher risk of complications. The infection rate was 30%. Malunion, chronic osteomyelitis and nonunion were observed in 14.5%, 12.3%, and 7% of the cases, respectively. More complications were observed with non-operative treatment (cast immobilization) than with surgical fixation. Although the surgical environment does not allow for internal fixation, poor results of non-operative management of open fractures should lead to the introduction of trainings on the proper use of external fixators. It is also advisable to support the development of locally produced external devices that utilize local source materials, which would make external fixation available at a reasonable cost.

Traumatic injuries of the distal tibiofibular syndesmosis.

The distal tibiofibular syndesmosis (DTFS) is frequently injured during ankle trauma. The sequelae can be significant, including chronic instability, early osteoarthritis and residual pain. The aim of this study is to summarize the current state knowledge about these injuries by answering four questions. They frequently occur in the context of an ankle sprain (20-40% of cases) or during various types of ankle fractures (20-100% of cases). They cannot be ruled out based solely on fracture type and must be investigated when a fibular or posterior malleolar fracture is present. Clinical examination and imaging are essential but do not provide a definitive diagnosis. Ultrasonography, CT scan and MRI have high sensitivity, but their static nature does not allow a treatment strategy to be defined. Dynamic radiographs must be taken, either with load or during a procedure. If instability is detected, stabilization is the general rule. In fracture cases, reduction is achieved by restoring the length and rotation of the distal fibular fragment, preferably during an open procedure. In sprain cases, reduction is not a problem unless there is ligament interposition. Tibiofibular fixation is done 1.5 to 3cm from the talocrural joint, while ensuring the reduction is perfect. The main complication-non-healing of the syndesmosis-is attributed to poor initial reduction. This or functional discomfort during weight bearing will require removal of the fixation hardware. In most cases, this allows functional recovery and correction of the inadequate reduction. Persistence of instability will require ligament reconstruction or fusion of the syndesmosis. Chronic instability can lead to ankle osteoarthritis. LEVEL OF EVIDENCE: V, expert opinion.

Can infection be predicted after intramedullary nailing of tibial shaft fractures?

Despite the progress in tibial fracture care, some patients contract infection following intramedullary nailing. We analyzed which risk factors could predict infection in 171 tibial fractures. The independent variables included age, gender, body mass index, and comorbidities, along with external factors of fracture pattern, nailing settings, and treatment processing time. A multiple logistic regression was used to identify infection risk factors. The risk of infection significantly increased according to the open grading, the fractures' classification, time until antibiotic administration, and time until nailing. Gustilo type I fractures presented a higher rate of infection than expected, explained by a longer delay before surgery. The probabilistic equation allows infection prediction with high sensitivity and specificity. In total, we showed that no antibiotics' prescription in emergency service and a transverse fracture pattern were predictors of infection. An infection risk score can be computed, aiding surgeons in decision making. Outcomes could improve keeping these observations in mind. Level of evidence: Retrospective cohort study. Level iii.

Predictors of mechanical complications after intramedullary nailing of tibial fractures.

INTRODUCTION: Intramedullary (IM) nailing is the gold standard treatment for tibial shaft fractures, but can be associated with various mechanical complications, including delayed union. HYPOTHESIS: We believe that complications do not occur randomly, but in certain conditions that contribute to their development. Risk factors likely to predict delayed union can be identified to support prevention. MATERIALS AND METHODS: A cohort of 171 fractures treated by IM nailing between 2005 and 2015 was reviewed retrospectively. Independent variables included intrinsic, patient-related factors and extrinsic factors such as those related to the fracture or surgery. A multiple logistic regression model was used to determine which factors can predict each type of complication. RESULTS: Delayed union occurred in 22.8% of patients. Smoking and high-energy trauma were risk factors. Hardware breakage was significantly reduced (p<0.05) when the nail diameter was greater than 10mm. A nail diameter/reamer diameter ratio outside the recommended limits (0.80-0.99) was more likely to be associated with screw failure. Diabetes is a risk factor for hardware migration, which itself is associated with other complications. DISCUSSION: Nonunion is the most common complication after IM nailing of tibial shaft fractures. Smoking cessation after a fracture is necessary in our opinion, even if the literature is ambivalent on this aspect and stopping to smoke once the fracture occurs may not be sufficient to prevent a poor outcome. Use of a nail diameter/reamer diameter between 0.80 and 0.99 favors union and prevents hardware breakage. Hardware migration in a diabetic patient may be a warning sign of other types of complications. LEVEL OF EVIDENCE: Retrospective cohort study. Level IV.