Optimized Workflow for High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to pathological alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto ITO coated glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracks, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 μm) MALDI IMS was leveraged to characterize lipid distributions in fresh-frozen bone, and complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Furthermore, we discovered that [sphingomyelin(42:1) + H]+ was abundant in megakaryocytes, whereas [sphingomyelin(42:2) + H]+ was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Therefore, this application of multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage in a preserved native bone microenvironment.

Performance of cooling materials and their composites in maintaining freezing temperature during irradiation and transportation of bone allografts

Purpose: Bone allografts supplied by University Malaya Medical Centre Bone Bank are sterilized by gamma radiation at 25 kGy in dry ice (DI) to minimize radiation effects. Use of cheaper and easily available cooling materials, gel ice (GI) and ice pack (IP), was explored. Composites of DI and GI were also studied for the use in routine transportations and radiation process. Methods: (a) Five dummy bones were packed with DI, GI, or IP in a polystyrene box. The bone temperatures were monitored while the boxes were placed at room temperature over 96 h. Durations for each cooling material maintaining freezing temperatures below −40°C, −20°C, and 0°C were obtained from the bone temperature over time profiles. (b) Composites of DI (20, 15, 10, 5, and 0 kg) and GI were used to pack five dummy bones in a polystyrene box. The durations maintaining varying levels of freezing temperature were compared. Results: DI (20 kg) maintained temperature below −40°C for 76.4 h as compared to 6.3 h in GI (20 bags) and 4.0 h in IP (15 packs). Composites of 15DI (15 kg DI and 9 GI bags) and 10DI (10 kg DI and 17 GI bags) maintained the temperature below −40°C for 61 and 35.5 h, respectively. Conclusion: Composites of DI and GI can be used to maintain bones in deep frozen state during irradiation, thus avoiding radiation effects on biomechanical properties. Sterile frozen bone allograft with preserved functional properties is required in clinical applications.

Mandibular Reconstruction by Using a Liquid Nitrogen-Treated Autograft in a Dog with an Oral Tumor

ABSTRACT A 10 yr old intact female German shepherd dog presented with a large peripheral odontogenic fibroma and malignant melanoma on her lower jaw. The tumor was resected with a unilateral subtotal rostral hemimandibulectomy. After the mandible was removed, it was devitalized intraoperatively by freezing it in liquid nitrogen. It was subsequently reimplanted. New bone tissue formed in the gap between the frozen bone and the host bone. The regenerated bone contained osteocytes, osteoblasts, and blood vessels. The cosmetic appearance of the dog was preserved. The dog had normal mastication. The malignant melanoma recurred rostral of the left canine tooth at 159 days after the reconstruction surgery. A subtotal hemimandibulectomy was consequently performed. This is the first reported case of mandibular reconstruction using a liquid nitrogen-treated autograft in a dog with oral tumors.

Volumetric Stability of Fresh Frozen Bone Blocks in Atrophic Posterior Mandible Augmentation

Fresh frozen bone allografts (FFB) have become an alternative for bone augmentation in the past decades, especially because of the absence of recent reports of disease transmission or immunologic reactions when it is used. The aim of this prospective controlled study is to evaluate volumetric changes of newly created bone following reconstruction of the atrophic posterior mandible. Twenty consecutive patients presenting for reconstruction of posterior mandibular alveolar bone ridge width ≤6.0 mm and/or height ≤6.0 who met all inclusion and exclusion criteria were included. FFB blocks were used. The main outcome variable investigated was bone volume dynamics. Vertical, horizontal, and 3-dimensional bone gain data were measured from computerized tomography scans. The main predictor variable was time evaluated at 3 points: immediately after surgery (T1), at implant placement (T2), and 1 year after functional loading (T3). Secondary outcome parameters evaluated were implant survival, histologic findings, and microtomographic morphometry. The study included 28 hemi-mandibles, 50 FFB bone blocks, and 15 female and 5 male patients (mean age, 51.8 years). Block and implant survival rates were 100% and 96%, respectively, after 31.75 months of follow-up. Vertical and horizontal bone gain at T2 was 5.15 and 6.42 mm, respectively. Volumetric resorption was 31% at T2, followed by an additional 10% reduction at T3. Histologic evaluation showed newly formed vital bone in intimate contact with the remaining FFB. Microtomography revealed 31.8% newly formed bone, 14.5% remaining grafted bone, and 53.7% connective tissue and bone marrow. Thus, FFB blocks may lead to new bone formation and consolidation, with satisfactory volumetric bone maintenance, allowing implant-supported rehabilitation with high success rates.