Partial enzyme digestion facilitates regeneration of crushed nerve in rat.

Peripheral nerve injury is a life-changing disability with significant socioeconomic consequences. In this rat model, we propose that partial enzyme digestion can facilitate the functional recovery of a crushed nerve. The sciatic nerves were harvested and in vitro cultured with the addition of Liberase to determine the appropriate enzyme amount in the hyaluronic acid (HA) membrane. Then, the sciatic nerve of adult male Sprague-Dawley rats was exposed, crushed, and then treated with partial enzyme digestion (either 0.001 or 0.002 unit/mm2 Liberase-HA membrane). The sciatic function index (SFI) for functional recovery of the sciatic nerve was evaluated. After 2 h of in vitro digestion, fascicles and axons were separated from each other, with the cells mobilized. Greater destruction of histology structures occurred in the high enzyme (Liberase-HA membrane at 0.002 unit/mm2) group at 24 h than in the low enzyme (0.001 unit/mm2) group at 48 h. In the SFI evaluation, the improvement in 0.001 unit/mm2 Liberase group was significantly better than control and 0.002 unit/mm2 Liberase group. Our study demonstrated that appropriate enzyme digestion had a significantly faster and earlier recovery.

Collagenase treatment of cartilaginous matrix promotes fusion of adjacent cartilage.

In articular cartilage-repair, grafts usually fuse unsatisfactorily with surrounding host cartilage. Enzymatic dissociation of cartilaginous matrix to free chondrocytes may benefit fusion. We tested such a hypothesis with human cartilage in vitro, and with porcine cartilage in vivo. Human articular cartilage was collected from knee surgeries, cut into disc-and-ring sets, and randomly distributed into three groups: disc-and-ring sets in Group 1 were left untreated; in Group 2 only discs, and in Group 3 both discs and rings were treated with enzyme. Each disc-and-ring reassembly was cultured in a perfusion system for 14 days; expression of cartilage marker proteins and genes was evaluated by immunohistochemistry and PCR. Porcine articular cartilage from knees was similarly fashioned into disc-and-ring combinations. Specimens were randomly distributed into a control group without further treatment, and an experimental group with both disc and ring treated with enzyme. Each disc-and-ring reassembly was transplanted into subcutaneous space of a nude mouse for 30 days, and retrieved to examine disc-ring interface. In in vitro study with human cartilage, a visible gap remained at disc-ring interfaces in Group 1, yet became indiscernible in Group 2 and 3. Marker genes, including type II collagen, aggrecan and Sox 9, were well expressed by chondrocytes in all specimens, indicating that chondrocytes' phenotype retained regardless of enzymatic treatment. Similar results were found inin vivo study with porcine cartilage. Enzymatic dissociation of cartilaginous matrix promotes fusion of adjacent cartilage. The clinical relevance may be a novel method to facilitate integration of repaired cartilage in joints.

Percutaneous pedicle screw placement under single dimensional fluoroscopy with a designed pedicle finder-a technical note and case series.

BACKGROUND CONTEXT: Minimally invasive spine surgery has become increasingly popular in clinical practice, and it offers patients the potential benefits of reduced blood loss, wound pain, and infection risk, and it also diminishes the loss of working time and length of hospital stay. However, surgeons require more intraoperative fluoroscopy and ionizing radiation exposure during minimally invasive spine surgery for localization, especially for guidance in instrumentation placement. In addition, computer navigation is not accessible in some facility-limited institutions. PURPOSE: This study aimed to demonstrate a method for percutaneous screws placement using only the anterior-posterior (AP) trajectory of intraoperative fluoroscopy. STUDY DESIGN: A technical report (a retrospective and prospective case series) was carried out. PATIENT SAMPLE: Patients who received posterior fixation with percutaneous pedicle screws for thoracolumbar degenerative disease or trauma comprised the patient sample. METHOD: We retrospectively reviewed the charts of consecutive 670 patients who received 4,072 pedicle screws between December 2010 and August 2015. Another case series study was conducted prospectively in three additional hospitals, and 88 consecutive patients with 413 pedicle screws were enrolled from February 2014 to July 2016. The fluoroscopy shot number and radiation dose were recorded. In the prospective study, 78 patients with 371 screws received computed tomography at 3 months postoperatively to evaluate the fusion condition and screw positions. RESULTS: In the retrospective series, the placement of a percutaneous screw required 5.1 shots (2-14, standard deviation [SD]=2.366) of AP fluoroscopy. One screw was revised because of a medialwall breach of the pedicle. In the prospective series, 5.8 shots (2-16, SD=2.669) were required forone percutaneous pedicle screw placement. There were two screws with a Grade 1 breach (8.6%), both at the lateral wall of the pedicle, out of 23 screws placed at the thoracic spine at T9-T12. Forthe lumbar and sacral areas, there were 15 Grade 1 breaches (4.3%), 1 Grade 2 breach (0.3%), and 1 Grade 3 breach (0.3%). No revision surgery was necessary. CONCLUSION: This method avoids lateral shots of fluoroscopy during screw placement and thus decreases the operation time and exposes surgeons to less radiation. At the same time, compared with the computer-navigated procedure, it is less facility-demanding, and provides satisfactory reliability and accuracy.

The effect of annular repair on the failure strength of the porcine lumbar disc after needle puncture and punch injury.

PURPOSE: The purpose was to quantify the structural integrity of annulus fibrosis (AF) after injuries with repair. METHODS: Punctures in the AF of 6-month-old porcine spine specimens were made with 18-, 20-, 22-, 24-, and 26-gauge needles. Leakage testing was performed immediately after needle puncture (Group 1), after the puncture was repaired with a modified purse-string suture (MPSS) (Group 2), and after needle puncture with immediate repair (Group 3). Punch injuries repaired with the MPSS alone, or with an AF graft and MPSS were also examined. RESULTS: There was no leakage from 26-gauge needle punctures. Pressures at which the nucleus pulposus leaked from the 24-, 22-, 20- and 18-gauge needle punctures (Group 1) were 4.28, 2.03, 1.27, and 1.06 MPa, respectively. Failure pressure after repair (Group 2, 3) was significantly greater than without (Group 1). Failure pressure in Group 3 was much greater than in Group 2 with 18- and 20-gauge punctures. Punch injury repaired with a graft and MPSS had significantly greater failure pressure than repair with MPSS alone (1.88 vs. 1.02 MPa, p = 0.0001). CONCLUSIONS: The MPSS can restore the mechanical integrity of the AF after needle puncture. An annular graft along with a MPSS may increase the structural integrity of the AF after a punch injury. As this was an acute animal study, the measurements and results may not directly translate to the human intervertebral disc.

Reinforced bioresorbable implants for craniomaxillofacial osteosynthesis in pigs.

Our aim was to design a new bioresorbable fixation device for craniomaxillofacial surgery based on reinforcement of fibres between 2 different polymers. The final device, the 2.8 mm polyglycolide fibre-reinforced-poly-l-lactide screw (PGA FR-PLLA; PLLA/PGA: 70%:30%), was evaluated for its mechanical properties and compared with a commercial resorbable device that was not reinforced with fibre. To model clinical conditions, a unilateral sagittal split ramus osteotomy fixation model in pigs was then used to compare the clinical effectiveness of the resorbable screw with that of a titanium screw in vivo, followed by 3-dimensional reconstructive imaging and histological analysis. Finally, an ex vivo biomechanical test was completed to investigate the immediate fixation stability of the newly designed screws. The PGA FR-PLLA screw resulted in clinical healing that was comparable to that of the titanium screw and was mechanically superior to the commercial device, indicating that the newly-developed screws have a potential clinical application.

The effect of a new anular repair after discectomy in intervertebral disc degeneration: an experimental study using a porcine spine model.

STUDY DESIGN: In vitro and in vivo studies to assess the effect of direct anular repair on subsequent degeneration of intervertebral discs (IVDs). OBJECTIVE: To assess whether a new suturing method could provide sealing effect on IVD after discectomy, and influence degenerative process of IVD. SUMMARY OF BACKGROUND DATA: Recurrent disc herniation and subsequent disc degeneration are major problems after discectomy. Anular repair can reduce the risk of recurrence, but its effect on disc degeneration needs more investigation. METHODS: A new suturing technique, the modified purse-string suture (MPSS), was designed for direct closure of anular incision. Intact motion segments of porcine lumbar spine were used to validate this technique in resisting disc pressure under mechanical loadings. A transverse slit incision was made in the anterior anulus of porcine cervical discs, with or without sealing of the anular defect by this suturing method. Magnetic resonance imaging grading was recorded before and after surgery. Anular healing was assessed histologically and gene expression of aggrecan, collagen type I, II, and matrix metalloproteinase-13 in nucleus pulposus were investigated. RESULTS: The average failure force of axial compression was 1150.3 ± 121.1 N for a simple suture, and 2917.9 ± 627.6 N for a MPSS. Cyclic loading test showed that the repaired discs succeeded against repeated compression forces. Magnetic resonance imaging and gross appearances showed lesser degenerative changes in repaired discs than in injured discs at each time period. In repaired discs, mRNA expression of aggrecan and type II collagen downregulated slightly with time, whereas it decreased rapidly and persistently in unrepaired discs. Histologic findings showed primary healing of outer anular tract in repaired discs. CONCLUSION: In this pilot study, the MPSS can provide effectively sealing for damaged anulus to withstand stresses. Direct repair of anular incision by this suturing method does significantly slow down degenerative process within discs after discectomy.

Clinical result of sintered bovine hydroxyapatite bone substitute: analysis of the interface reaction between tissue and bone substitute.

BACKGROUND: Autogenic bone graft is the first choice for managing bone defects. However, donor site-associated morbidity and limited bone volume are constraints in clinical applications. Allografts can provide sufficient amounts for bone defects but have a high risk of infection. Bone substitute composed of hydroxyapatite (HA) is an alternative material for avoiding the aforementioned risks. Sintered bovine bone is a naturally occurring HA that has been proved to have excellent bioactivity for inducing osteoblastic expression and new bone formation in animal studies. The objective of this study was to evaluate the interactions between the tissue and the bone substitute composed of HA (sintered from bovine bone) in the human body. METHODS: From 2003 to 2005, a total of 33 patients were enrolled to receive the sintered bovine HA as a bone substitute. Inclusion criteria were fractures with bony defects, benign bone tumors with a cavity, and spinal fusions. Bone healing was monitored by a series of radiographs, and bone microstructure was checked by scanning electron microscopy (SEM) and von Kossa staining. RESULTS: In 81.8% (27/33) of cases, significant fusion mass formation was visible in the radiographs after 6-12 months. New bone formation on the surface of the sintered bovine HA was seen under microscopic observation. Tight bonding between the interface of the bone and the sintered bovine HA was shown with SEM/energy-dispersive spectroscopy and von Kossa staining. CONCLUSIONS: Sintered bovine HA is a suitable material as a bone substitute to provide bone growth and promote bone healing.

Comparison of articular cartilage repair by autologous chondrocytes with and without in vitro cultivation.

OBJECTIVE: autologous chondrocyte implantation usually requires in vitro cell expansion before implantation. We compared the efficacy of cartilage regeneration by in vitro-expanded chondrocytes at high density and freshly harvested chondrocytes at low density. DESIGN: surgically created osteochondral defects at weight-bearing surface of femoral condyles of domestic pigs were repaired by biphasic cylindrical porous plugs of DL-poly-lactide-co-glycolide and beta-tricalcium phosphate. Plugs were seeded with autologous chondrocytes in its chondral phase, and press-fit to defects. Seeded cells were (1) in vitro-expanded chondrocytes harvested from stifle joint 3 weeks before implantation and (2) freshly harvested chondrocytes from recipient knee. Seeding densities were 70 x 10(6) and 7 x 10(6) cells/mL, respectively. Cell-free plugs served as control and defects remained untreated as null control. Outcome was examined at 6 months with International Cartilage Repair Society Scale. RESULTS: the two experimental groups were repaired by hyaline cartilage with collagen type II and Safranin-O. Tissue in control group was primarily fibrocartilage. No regeneration was found in null control. Experimental groups had higher mean International Cartilage Repair Society scores than control in surface, matrix, and cell distribution, but were comparable with control in cell viability, subchondral bone, and mineralization. No significant difference existed between two experimental groups in any of the six categories. Uni-axial indentation test revealed similar creeping stress-relaxation property as native cartilage on experimental, but not control, specimen. CONCLUSIONS: cartilage could regenerate in both experimental models, in comparable quality. Culture of chondrocytes before implantation is not necessary.

Chondrocytes culture in three-dimensional porous alginate scaffolds enhanced cell proliferation, matrix synthesis and gene expression.

For the limited availability of autologous chondrocytes, a cultured system for expansion in vitro until sufficient cells are obtained must be developed. These cells must maintain their chondrocyte phenotype in vitro as well as in vivo, following implantation to ensure that differentiated chondrocytes synthesize a normal hyaline cartilage matrix and not a fibro-cartilage matrix. This study uses porous three-dimensional (3-D) alginate scaffolds within a perfusion system to culture low-density (5 x 10(5) cells) primary porcine chondrocytes for 1-4 weeks to study their proliferation and differentiation. The results of RT-PCR reveal that most cells could maintain their differentiation state for up to 4 weeks of culturing. Chondrocytes proliferated to 3 x 10(7) cells after 4 weeks in culture. Alginate scaffolds induced the formation of chondrocyte clusters and stimulated the synthesis of matrix, which effects were evaluated using histology and electron microscopy. These findings demonstrate that culturing chondrocytes in alginate scaffolds may effectively prevent the dedifferentiation and improve autologous chondrocyte transplantation.

Use of porous alginate sponges for substantial chondrocyte expansion and matrix production: effects of seeding density.

Autologous cell transplantation is a promising approach for cartilage repair, but the expansion of chondrocytes in a monolayer, a common approach to amplifying the cell number, inevitably leads to cell de-differentiation. To explore whether porous alginate sponges could be utilized for chondrocyte expansion and investigate the effects of seeding densities, the porcine chondrocytes were seeded to porous alginate sponges at low (5 x 10(5) cells per 40 sponges), medium (5 x 10(6) cells per 40 sponges), or high (2 x 10(7) cells per 40 sponges) density. After 4-week perfusion culture, all three groups resulted in chondrocyte proliferation, maintenance of chondrocytic gene (collagen II, Sox 9 and aggrecan) expression, and formation of cell clusters resembling cartilaginous tissues. The higher the seeding density, the higher the final cell density and GAGs production and, accordingly, the larger the cell clusters. Strikingly, the cumulative expansion ratios achieved by the low-density group ( approximately 150-fold) significantly exceeded those achieved by the medium (approximately 21-fold) and high (approximately 4.7-fold) density groups, as well as those achieved using other scaffolds. In conclusion, seeding chondrocytes to the alginate sponges at a low density, combined with perfusion culture, represents a drastic improvement in expanding autologous chondrocytes.

Repair of porcine articular cartilage defect with a biphasic osteochondral composite.

Autologous chondrocyte implantation (ACI) has been recently used to treat cartilage defects. Partly because of the success of mosaicplasty, a procedure that involves the implantation of native osteochondral plugs, it is of potential significance to consider the application of ACI in the form of biphasic osteochondral composites. To test the clinical applicability of such composite construct, we repaired osteochondral defect with ACI at low cell-seeding density on a biphasic scaffold, and combined graft harvest and implantation in a single surgery. We fabricated a biphasic cylindrical porous plug of DL-poly-lactide-co-glycolide, with its lower body impregnated with beta-tricalcium phosphate as the osseous phase. Osteochondral defects were surgically created at the weight-bearing surface of femoral condyles of Lee-Sung mini-pigs. Autologous chondrocytes isolated from the cartilage were seeded into the upper, chondral phase of the plug, which was inserted by press-fitting to fill the defect. Defects treated with cell-free plugs served as control. Outcome of repair was examined 6 months after surgery. In the osseous phase, the biomaterial retained in the center and cancellous bone formed in the periphery, integrating well with native subchondral bone with extensive remodeling, as depicted on X-ray roentgenography by higher radiolucency. In the chondral phase, collagen type II immunohistochemistry and Safranin O histological staining showed hyaline cartilage regeneration in the experimental group, whereas only fibrous tissue formed in the control group. On the International Cartilage Repair Society Scale, the experimental group had higher mean scores in surface, matrix, cell distribution, and cell viability than control, but was comparable with the control group in subchondral bone and mineralization. Tensile stress-relaxation behavior determined by uni-axial indentation test revealed similar creep property between the surface of the experimental specimen and native cartilage, but not the control specimen. Implanted autologous chondrocytes could survive and could yield hyaline-like cartilage in vivo in the biphasic biomaterial construct. Pre-seeding of osteogenic cells did not appear to be necessary to regenerate subchondral bone.

Injecting partially digested cartilage fragments into a biphasic scaffold to generate osteochondral composites in a nude mice model.

This study proposed a novel scaffold with heterogeneous morphology that mimics the natural tissue. Its upper part contains a hollow cavity surrounded by a wall of poly(L-lactic-co-glycolic acid) (PLGA) porous membrane for injecting cartilage tissue and cells. An interconnecting porous structure located under the hollow cavity was made of composite materials that combined PLGA and beta-tricalcium phosphate (beta-TCP) to simulate the subchondral bone. Adult pig articular cartilage was cut and sieved into small fragments. The tissue fragments was partially digested by 0.1% collagenase for 0, 2, 4, and 6 h and injected into the hollow cavity of the biphasic scaffold. The biphasic scaffolds were then implanted into the subcutaneous pocket of nude mice for 4 weeks. No tissue bonding or new cartilaginous tissue formation was identified in the cartilage fragment without enzymatic treatment. The cartilage fragments digested with 2 h of collagenase digestion were partially integrated after implantation. The integrative properties of the cartilage fragment depended on the extent of enzymatic digestion. Releasing cells at the tissue surface enhanced confluence and bonding of the cartilage fragment matrix. Complete integration of the cartilage fragments and cartilage remodeling were achieved by digestion of the tissue fragments with 4 h of enzymatic treatment. The neocartilage grew from the upper hollow cavity into the lower PLGA/beta-TCP porous structure, forming an interface similar to that formed between cartilage and subchondral bone. This study combined the osteochondral scaffold and limited cartilage tissues to generate cartilage tissue in vivo intending for repairing full-thickness articular cartilage defects.

A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage.

A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.

Fabrication of porous biodegradable polymer scaffolds using a solvent merging/particulate leaching method.

This study developed a solvent merging/particulate leaching method for preparing three-dimensional porous scaffolds. Poly(L-lactic-co-glycolic acid) (PLGA) and sodium chloride particles were dry-mixed and cast into a special mold, through which a liquid could pass due to a pressure difference. An organic solvent was then poured into the mold to dissolve and merge the PLGA particles under negative pressure. A nonsolvent was conducted into the PLGA/salt composite to solidify and precipitate the merged PLGA matrix. Finally, a large amount of water was passed through the mold to leach out the salt particles so as to create a porous structure. The results revealed that a highly porous three-dimensional scaffold (>85 vol %) with a well interconnected porous structure could be achieved by this process. Porosity and the pore size of the scaffold were controlled using the ratio and the particle size of the added salt particles. A larger-volume scaffold was produced using a larger mold. This work provides a continuous and simple procedure for fabricating a bulk three-dimensional porous scaffold for tissue engineering.