The effects of prolonged deep freezing on the biomechanical properties of osteochondral allografts

Musculo-skeletal allografts sterilized and deep frozen are among the most common human tissue to be preserved and utilized in modern medicine. The effects of a long deep freezing period on cortical bone has already been evaluated and found to be insignificant. However, there are no reports about the influences of a protracted deep freezing period on osteochondral allografts. One hundred osteochondral cylinders were taken from a fresh specimen and humeral heads of 1 year, 2 years, 3 years and 4 year old bones. Twenty chips from each period, with a minimum of 3 chips per humeral head. Each was mechanically tested by 3 point compression. The fresh osteochondral allografts were significantly mechanically better than the deep frozen osteochondral allografts. There was no statistical significant time dependent difference between the deep frozen groups in relation to the freezing period. Therefore, we conclude that, from the mechanical point of view deep freezing of osteochondral allografts over a period of 4 years, is safe without further deterioration of the biomechanical properties of the osteochondral allografts.     

Repeated freeze–thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons

Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze–thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze–thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze–thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze–thaw cycles. In patella tendons, the ultimate stress, Young’s modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze–thaw cycles. In conclusion, we identified that cells surviving after freeze–thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze–thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity.     

Mechanical properties of radiation-sterilised human Bone-Tendon-Bone grafts preserved by different methods

Patellar tendon auto- and allo-grafts are commonly used in orthopedic surgery for reconstruction of the anterior cruciate ligaments (ACL). Autografts are mainly used for primary reconstruction, while allografts are useful for revision surgery. To avoid the risk of infectious disease transmission allografts should be radiation-sterilised. As radiation-sterilisation supposedly decreases the mechanical strength of tendon it is important to establish methods of allograft preservation and sterilisation assuring the best quality of grafts and their safety at the same time. Therefore, the purpose of this study was to compare the tensile strength of human patellar tendon (cut out as for ACL reconstruction), preserved by various methods (deep fresh freezing, glycerolisation, lyophilisation) and subsequently radiation-sterilised with doses of 0, 25, 50 or 100 kGy. Bone-Tendon-Bone grafts (BTB) were prepared from cadaveric human patella tendons with both patellar and tibial attachments. BTB grafts were preserved by deep freezing, glycerolisation or lyophilisation and were subsequently radiation-sterilised with doses of 0 (control), 25, 50 or 100 kGy. All samples were subjected to mechanical failure tensile tests with the use of Instron system in order to estimate their mechanical properties. All lyophilised grafts were rehydrated before performing of those tests. Obtained mechanical tests results of examined grafts suggest that deep-frozen irradiated grafts retain their initial mechanical properties to an extent which does not exclude their clinical application. All conducted experiments were approved by the Local Ethical Committee.     

Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics

Gamma irradiation from Cobalt 60 sources has been used to terminally sterilize bone allografts for many years. Gamma radiation adversely affects the mechanical and biological properties of bone allografts by degrading the collagen in bone matrix. Specifically, gamma rays split polypeptide chains. In wet specimens irradiation causes release of free radicals via radiolysis of water molecules that induces cross-linking reactions in collagen molecules. These effects are dose dependent and give rise to a dose-dependent decrease in mechanical properties of allograft bone when gamma dose is increased above 25 kGy for cortical bone or 60 kGy for cancellous bone. But at doses between 0 and 25 kGy (standard dose), a clear relationship between gamma dose and mechanical properties has yet to be established. In addition, the effects of gamma radiation on graft remodelling have not been intensively investigated. There is evidence that the activity of osteoclasts is reduced when they are cultured onto irradiated bone slices, that peroxidation of marrow fat increases apoptosis of osteoblasts; and that bacterial products remain after irradiation and induce inflammatory bone resorption following macrophage activation. These effects need considerably more investigation to establish their relevance to clinical outcomes. International consensus on an optimum dose of radiation has not been achieved due to a wide range of confounding variables and individual decisions by tissue banks. This has resulted in the application of doses ranging from 15 to 35 kGy. Here, we provide a critical review on the effects of gamma irradiation on the mechanical and biological properties of allograft bone.     

Radioprotection provides functional mechanics but delays healing of irradiated tendon allografts after ACL reconstruction in sheep

Successful protection of tissue properties against ionizing radiation effects could allow its use for terminal sterilization of musculoskeletal allografts. In this study we functionally evaluate Achilles tendon allografts processed with a previously developed radioprotective treatment based on (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) crosslinking and free radical scavenging using ascorbate and riboflavin, for ovine anterior cruciate ligament reconstruction. Arthroscopic anterior cruciate ligament (ACL) reconstruction was performed using double looped allografts, while comparing radioprotected irradiated and fresh frozen allografts after 12 and 24 weeks post-implantation, and to control irradiated grafts after 12 weeks. Radioprotection was successful at preserving early subfailure mechanical properties comparable to fresh frozen allografts. Twelve week graft stiffness and anterior-tibial (A-T) translation for radioprotected and fresh frozen allografts were comparable at 30 % of native stiffness, and 4.6 and 5 times native A-T translation, respectively. Fresh frozen allograft possessed the greatest 24 week peak load at 840 N and stiffness at 177 N/mm. Histological evidence suggested a delay in tendon to bone healing for radioprotected allografts, which was reflected in mechanical properties. There was no evidence that radioprotective treatment inhibited intra-articular graft healing. This specific radioprotective method cannot be recommended for ACL reconstruction allografts, and data suggest that future efforts to improve allograft sterilization procedures should focus on modifying or eliminating the pre-crosslinking procedure.     

The effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bone

The effects of cryopreservation on tendon allograft have been reported, but remain unclear, particularly the potential effects on mechanical properties and histological changes by ice crystal formation. There are also few studies about effects of heating for sterilization of tendon. We evaluated the effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bones (BTBs). BTBs were processed by cryopreservation at −80°C for 3 weeks, or heating at 80°C for 10 min. Tensile testing and histomorphological examination were performed. The cryopreservation of tendons showed less influences on their mechanical properties. When cryopreserved BTBs in frozen state were fixed by freeze-substitution method, many spaces were observed in interfibrillar substances. These results suggest that the collagen fibers of cryopreserved tendons were histomorphologically affected by ice crystals. The heating of tendons completely destroyed the collagen fibers of the tendons and is therefore thought to be inappropriate for the sterilization of BTBs.     

Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. Gamma irradiation is an effective method for providing terminal sterilization to biological tissue, but it is also reported to have deleterious effects on tissue mechanics in a dose-dependent manner. At irradiation ranges up to 25 kGy, a clear relationship between mechanical strength and dose has yet to be established. The aim of this study was to investigate the mechanical properties of bone and soft tissue allografts, irradiated on dry ice at a low absorbed dose (18.3–21.8 kGy) and a moderate absorbed dose (24.0–28.5 kGy), using conventional compressive and tensile testing, respectively. Bone grafts consisted of Cloward dowels and iliac crest wedges, while soft tissue grafts consisted of patellar tendons, anterior tibialis tendons, semitendinosus tendons, and fascia lata. There were no statistical differences in mechanical strength or modulus of elasticity for any graft irradiated at a low absorbed dose, compared to control groups. Also, bone allografts and two soft tissue allografts (anterior tibialis and semitendinosus tendon) that were irradiated at a moderate dose demonstrated similar strength and modulus of elasticity values to control groups. The results of this study support the use of low dose and moderate dose gamma irradiation of bone grafts. For soft tissue grafts, the results support the use of low dose irradiation.     

Solvent-dehydrated calvarial allografts in craniofacial surgery

Craniofacial surgery almost always requires the use of bone grafting. Although autografts are the standard procedure for bone grafting, it is sometimes not possible to harvest bone, and autografts have particular risks. The use of allograft bone provides a reasonable alternative to meet the need for graft material. Solvent dehydration is a multistage procedure in which human cadaveric bone is processed by osmotic exchange baths and gamma sterilization. This processing avoids the risk of infection transmission, decreases antigenicity, and does not weaken the mechanical properties of the bone. Solvent-dehydrated, gamma-irradiated human calvarial bone allografts were used for reconstruction of craniofacial deformities in 24 patients between 1988 and 2002. Resorption of the allografts and results of the surgical intervention were evaluated with plain radiographs and three-dimensional computed tomography 12 months after surgery, in 21 patients. Serologic tests for human immunodeficiency virus-1 antibody, hepatitis B surface antigen, and hepatitis C antigen were also performed. Biopsy specimens were taken from the allografts. Average follow-up in this group was 30 months (range, 8 to 60 months), and results of serologic tests were negative in all patients. Seventy-one percent of the patients (15 of 21) showed no resorption, with partial and complete allograft fusion. One patient had nearly total graft loss and the remaining five patients had 10 to 25 percent graft resorption. Rigid fixation of the allograft, contact with the dura and periosteum, and prevention of dead spaces around the allograft are the most important factors in achieving a satisfactory result. In solvent-dehydrated bone allografts, sterilization and antigenic tissue cleaning are achieved after several steps with a minimal dose of radiation. The result is a nonantigenic, sterile mechanical scaffold that can tolerate external forces. Although autografts are the standard in craniofacial surgery, solvent-dehydrated calvarial bone allografts produced successful results in selected cases.     

Effects of <Superscript>60</Superscript>Co gamma radiation dose on initial structural biomechanical properties of ovine bone—patellar tendon—bone allografts

Gamma radiation is established as a procedure for inactivating bacteria, fungal spores and viruses. Sterilization of soft tissue allografts with high dose ⁶⁰Co gamma radiation has been shown to have adverse effects on allograft biomechanical properties. In the current study, bone-patellar tendon-bone (BPTB) allografts from 32 mature sheep were divided into two treatment groups: low-dose radiation at 15 kGy (n = 16) and high-dose radiation at 25 kGy (n = 16) with the contralateral limb serving as a 0 kGy (n = 32) non-irradiated control. Half of the tendons from all treatment groups were biomechanically tested to determine bulk BPTB mechanical properties, cancellous bone compressive properties, and interference screw pull-out strength. The remaining tissues were prepared, implanted, and mechanically tested in an acute in vitro anterior crucial ligament (ACL) reconstruction. Low-dose radiation did not adversely affect mechanical properties of the tendon allograft, bone, or ACL reconstruction compared to internal non-irradiated control. However, high-dose radiation compromised bulk tendon load at failure and ultimate strength by 26.9 and 28.9%, respectively (P < 0.05), but demonstrated no negative effect on the cancellous bone compressive properties or interference screw pull-out strength. Our findings suggest that low dose radiation (15 kGy) does not compromise the mechanical integrity of the allograft tissue, yet high dose radiation (25 kGy) significantly alters the biomechanical integrity of the soft tissue constituent.     

Packin’ ya Eskies!

The objective was to document a Process Validation on the packaging of human tissue grafts using polystyrene boxes containing dry ice for short term storage. The aim was to give a high degree of assurance that the processed grafts would be maintained at −20°C for a period of time to allow distribution to customers. This study was designed to comply with the Australian GMP—Human Blood and Tissues and AATB Standards for Tissue Banking (Ed 12) American Association of Tissue Banks Section E4.141—Storage Conditions for Commonly Transplanted Human Tissue. Four Eskies were packed with 1, 4, 10 & 20 “dummy” allografts with thermocouples and Data Loggers attached with 3.5, 7, 15 and 20 kg of dry ice packed around the “dummy” allografts, respectively. All Eskies were weighed six times over a 48 h period and temperatures recorded. The results showed that one allograft in an Esky with 3.5 kg of dry ice was able to be stored for up to 31 h and fifteen allografts in an Esky containing 20 kg dry ice lasted 48 h.     

Effects of cell-seeding methods of human osteoblast culture on biomechanical properties of porous bioceramic scaffold

Many studies have been performed to accelerate osteoinduction and osteoconduction into porous ceramic scaffolds by seeding them with cells. In this study, we compared available cell-seeding methods on a porous β-tricalcium phosphate (β-TCP) scaffold and evaluated the effects of cell-seeding on the mechanical properties of the porous β-TCP scaffold. Three types of porous bioceramic scaffolds were used: dry scaffold, scaffold wetted with media, and scaffold cultivated with normal human osteoblasts (NHOs). Cell-seeding into the porous β-TCP scaffolds was performed by conventional, centrifuge, high-density, and vacuum methods. After confirming cell proliferation with MTT assay and cell staining, a compressive test was performed after 2 and 4 weeks of cell culture. The vacuum method based on the high-density cell culture inserted effectively NHOs into the β-TCP scaffolds. The compressive elastic modulus of wetted β-TCP scaffolds decreased significantly (p < 0.05) about 20∼30% after 2 and 4 weeks of incubation in comparison with that of the dry scaffold. However, the compressive strength of the scaffolds cultivated with NHOs for 3 weeks was significantly (p < 0.05) higher than that of scaffolds without NHOs. The vacuum with the high-density of cell-seeding seems to be a suitable method for seeding cells into complex porous ceramic scaffolds. Cell proliferation and uniform distribution in the scaffolds can change the initial mechanical properties of porous ceramic scaffolds.     

The restoration of full-thickness cartilage defects with mesenchymal stem cells (MSCs) loaded and cross-linked bilayer collagen scaffolds on rabbit model

Cartilage has a limited self-repair capability and the repair of large cartilage defects remains a challenge in clinic. This study aimed to investigate the effect of mesenchymal stem cells (MSCs) loaded three-dimensional bilayer collagen scaffold for cartilage repair. Cross-linked three-dimensional bilayer collagen scaffolds seeded with or without MSCs were implanted into large cartilage defects (4 mm in diameter; 3 mm in depth) in rabbit knees. The untreated cartilage defects served as control. The tissue response was evaluated at 6 and 12 weeks after implantation by general histology and semi-quantitative histological grading systems. In addition, the repaired tissues were evaluated by mechanical test at 12 weeks after implantation. The MSCs-loaded collagen scaffold group showed the most hyaline cartilage, highest histological scores and compressive modulus. Moreover, it showed a good integration with the subchondral bone and adjacent cartilage. The structure of the novel bilayer collagen scaffolds provided architectural support for the differentiation of MSCs and demonstrated successful induction of in vivo chondrogenesis. These findings suggested that MSCs-loaded bilayer collagen scaffold could be an appealing candidate to be used for cartilage regeneration.     

Fabrication of collagen hybridized elastic PLCL for tissue engineering

Biodegradable elastic poly(l-lactide-co-ε-caprolactone) (PLCL) (50:50) copolymer was blended with collagen (0.05, 0.1 and 0.2% w/w) in an acidic dioxane solution to form a collagen/PLCL hybrid material suitable for tissue engineering applications. Stability and dispersivity of collagen on collagen/PLCL hybrid films and collagen coated PLCL films under mechanical stress were determined by a collagen release test and water contact angle measurement. Hybrid films had a higher stability than collagen-coated PLCL films. Elastic recovery as well as high interconnectivity and uniform pore morphology of the hybrid scaffolds were not affected by the collagen concentration. Fibroblasts (NIH-3T3) cell culture test was performed for cell growth and viability evaluation. Collagen concentration had little affect on the initial cell adhesion after 4 h cell culture; but after 48 h cell culture, increased cell proliferation on the hybrid films was observed. The hybrid material can be applied as a scaffold for vessel and cartilage regeneration for mechano-active tissue engineering.     

In vitro Response of the Bone Marrow-Derived Mesenchymal Stem Cells Seeded in a Type-I Collagen-Glycosaminoglycan Scaffold for Skin Wound Repair Under the Mechanical Loading Condition

In order to achieve successful wound repair by regenerative tissue engineering using mesenchymal stem cells (MSCs), it is important to understand the response of stem cells in the scaffold matrix to mechanical stress.
To investigate the clinical effects of mechanical stress on the behavior of cells in scaffolds, bone marrow-derived mesenchymal stem cells (MSCs) were grown on a type-I collagen-glycosaminoglycan (GAG) scaffold matrix for one week under cyclic stretching loading conditions.
The porous collagen-GAG scaffold matrix for skin wound repair was prepared, the harvested canine MSCs were seeded on the scaffold, and cultured under three kinds of cyclic stretching loading conditions ( 0%: control, 5% strain, 15% strain ). After 7 days incubation, MSCs were evaluated histologically and immunohistochemically regarding the proliferation and differentiation.
Cultured MSCs in the high strain (15% strain) group showed activea-smooth muscle actin (α-SMA) expression and poor differentiation into type-I collagen-positive cells, whereas enhanced differentiation into type-I collagen positive cells and a lack ofa-SMA expression where shown in the lower stress (5% strain) group. These results suggest that mechanical stress may affect the proliferation and differentiation of stem cells, and subsequently the wound healing process, through attachment interactions between the stem cells and scaffold matrix. Our findings provide an additional consideration for clinical treatment of wound repair using regenerative tissue engineering.     

Local delivery of FTY720 accelerates cranial allograft incorporation and bone formation

Endogenous stem cell recruitment to the site of skeletal injury is key to enhanced osseous remodeling and neovascularization. To this end, this study utilized a novel bone allograft coating of poly(lactic-co-glycolic acid) (PLAGA) to sustain the release of FTY720, a selective agonist for sphingosine 1-phosphate (S1P) receptors, from calvarial allografts. Uncoated allografts, vehicle-coated, low dose FTY720 in PLAGA (1:200 w:w) and high dose FTY720 in PLAGA (1:40) were implanted into critical size calvarial bone defects. The ability of local FTY720 delivery to promote angiogenesis, maximize osteoinductivity and improve allograft incorporation by recruitment of bone progenitor cells from surrounding soft tissues and microcirculation was evaluated. FTY720 bioactivity after encapsulation and release was confirmed with sphingosine kinase 2 assays. HPLC-MS quantified about 50% loaded FTY720 release of the total encapsulated drug (4.5 μg) after 5 days. Following 2 weeks of defect healing, FTY720 delivery led to statistically significant increases in bone volumes compared to controls, with total bone volume increases for uncoated, coated, low FTY720 and high FTY720 of 5.98, 3.38, 7.2 and 8.9 mm³, respectively. The rate and extent of enhanced bone growth persisted through week 4 but, by week 8, increases in bone formation in FTY720 groups were no longer statistically significant. However, micro-computed tomography (microCT) of contrast enhanced vascular ingrowth (MICROFIL?) and histological analysis showed enhanced integration as well as directed bone growth in both high and low dose FTY720 groups compared to controls.     

Combined CXCR3/CCR5 blockade attenuates acute and chronic rejection

Chemokine-chemokine receptor interactions orchestrate mononuclear cells recruitment to the allograft, leading to acute and chronic rejection. Despite biologic redundancy, several experimental studies have demonstrated the importance of CXCR3 and CCR5 in acute rejection of allografts. In these studies, deficiency or blockade of CXCR3 or CCR5 led to prolongation of allograft survival, yet allografts were ultimately lost to acute rejection. Given the above findings and the specificity of mononuclear cells bearing CXCR3 and CCR5, we hypothesized that combined blockade of CXCR3 and CCR5 will lead to indefinite (>100 days) graft survival in a full MHC-mismatched murine cardiac allograft model. The donor hearts in the control group were rejected in 6 +/- 1 days after transplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival >15-fold vs the control group; all allografts survived for >100 days. More importantly, the donor hearts did not display any intimal lesions characteristic of chronic rejection. Further analysis of the donor hearts in the CXCR3/CCR5 blockade group demonstrated graft infiltration with CD4(+)CD25(+) T cells expressing the Foxp3 gene. Depletion of CD25(+) cells in the combined CXCR3 and CCR5 blockade group resulted in acute rejection of the allografts in 22 +/- 2 days. Combined CXCR3 and CCR5 blockade also reduced alloantigen-specific T lymphocyte proliferation. Combined CXCR3 and CCR5 blockade is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated, in part, by CD25(+) regulatory T cell recruitment and control of T lymphocyte proliferation.     

Preservation methods for the storage of anaerobic sludges

Different preservation methods were evaluated for the storage of anaerobic sludges: room temperature, refrigeration at 4 °C, freezing at –20 °C and freeze-drying. Specific methanogenic activity for glucose and acetate were used as indicators of the subsequent recovery of the anaerobic sludge. Storage at room temperature and refrigeration resulted in a better conservation of the methanogenic activity than freezing and freeze-drying.     

A novel model-gel-tissue assay analysis for comparing tumor elastic properties to collagen content

In previous work, a new assay was realized for determining soft-tissue mechanical properties. The method, named the model-gel-tissue (MGT) assay, couples material testing with a finite element model built from a micro-CT image acquisition of a gel-embedded tissue specimen to determine its mechanical properties. Given recent reports demonstrating that increased stromal collagen promotes mammary tumor initiation and proliferation, in this paper, the MGT assay is used to evaluate the modulus of murine mammary tumors and is subsequently correlated quantitatively to type I collagen content. In addition, preliminary testing of the assay sensitivity with respect to gel-volume to tissue-mass ratio is reported here. The results demonstrate a strong linear correlation between tumor mechanical properties and collagen content (R ² = 0.9462). This result is important because mechanical stiffness as provided by the MGT assay is very similar to parameters under clinical investigation using elastographic imaging techniques. The sensitivity tests indicated that an approximate gel-volume to tissue-mass ratio threshold of 16.5 ml g⁻¹ is needed for successful analysis. This is an important result in that it presents guideline constraints for conducting this analysis.     

Long-term preservation of yeast cultures in liquid nitrogen

Nineteen strains of taxonomieally diverse yeast species tested survived freezing and subsequent five-year storage in liquid nitrogen at ™196 °C, using a medium M 2 composed of malt extract, yeast extract, peptone, calf serum and dimethyl sulfoxide. Viability of the yeast cultures after long-term storage ranged from 5 to 97 % (average 62 %) compared with the viability of the cultures prior to freezing. The use of liquid nitrogen refrigeration for preserving yeast cultures is strongly advocated.