Preparing recombinant "Split AEP" for protein labeling.

A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity. Here, we report a novel preparation protocol, in which the cap and catalytically active core domains are produced as separate entities. The active enzyme can be obtained in two chromatographic steps, immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC), with no acid activation required, thereby shortening the purification procedure from at least 2 days to less than 6 h. In addition to the original C247A mutation which enhanced reaction with various amino nucleophiles, an extra D29E mutation was introduced to prevent self-cleavage, which led to noticeable improvements in homogeneity and activity of the enzyme. Indeed, the resulting "split AEP" (i.e., core domain of OaAEP1-D29E/C247A) exhibited improved catalytic efficiency constant (kcat/KM) that was found to be ∼3-fold higher than that of the original acid-activated counterpart (OaAEP1-C247A). Furthermore, we described a protein labeling protocol that couples the enzymatic reaction with an irreversible chemical transformation, thereby enabling high conversion of labeled protein with a lowered amount of reagent. Precisely, an alternative Asn-Cys-Leu (NCL) recognition sequence was used for substrate recognition. As the byproduct contains an N-terminal cysteine, it can be transformed into an inert 1,2 aminothiol motif by reacting with formylphenyl boronic acid (FPBA). Finally, the opportunities and challenges associated with the use of asparaginyl ligase are discussed.

The differential mediating effects of pain and depression on the physical and mental dimension of quality of life in Hong Kong Chinese adults.

OBJECTIVE: The impact of pain and depression on health-related quality of life (QoL) is widely investigated, yet the pain-depression interactions on QoL remain unclear. This study aims to examine the pain-depression-QoL mediation link. METHODS: Pain severity were assessed in a sample of Chinese professional teachers (n = 385). The subjects were also assessed on depressive symptoms and QoL. Regression models were fitted to evaluate the pain-depression-QoL relationships. RESULTS: About 44% of the sample had 3-5 painful areas in the past 3 months. Shoulder pain (60%) and headache (53%) were common painful areas. The results of regression analyses showed that pain mediated the effects of depression on the mental aspect of QoL (standardized beta = -0.111; Sobel test: z = -3.124, p < 0.005) whereas depression mediated the effects of pain on the physical aspect of QoL (standardized beta = -0.026; Sobel test: z = -4.045, p < 0.001). CONCLUSIONS: Our study offered tentative evidence that pain and depression impacted differently on the mental and physical aspect of QoL. As these findings were based on a Chinese teacher sample, future studies should employ more representative samples across cultures to verify the present data.

[Bioluminescent reporter genes]. / Systemy genów reporterowych opartych na zjawisku bioluminescencji.

Reporter genes typically are used to monitor changes in transcriptional rate, which can vary quickly in response to a specific cellular event. Here we give background on bioluminescent reporters and assays and their uses in research.

Toward a structural understanding of the dehydratase mechanism.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.

Screening trial with the coordinated gold compound auranofin using mouse lymphocyte leukemia P388.

The coordinated gold compound, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S-triethylphosphine-gold (auranofin) was found to be effective in increasing the life span of C57BL x DBA/2 F1 mice inoculated with the lymphocytic leukemia P388. A number of dose schedules were used, the lowest dose being 6 mg/kg every fourth day and the highest dose being 6.0 mg/kg twice daily for 9 days; the lowest and highest doses produced treated versus control ratios of 140 and 220%, respectively. All treatment groups achieved the minimum treated versus control ratio of 125%. Animal weights remained stable at twice-daily and high-dose-daily regimens. Increased life span and weight changes were both found to correlate with drug concentration and/or dose frequency.

The structure at 1.7 A resolution of the protein product of the At2g17340 gene from Arabidopsis thaliana.

The crystal structure of the At2g17340 protein from A. thaliana was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 16.9% (Rfree = 22.1%) at 1.7 A resolution. At2g17340 is a member of the Pfam01937.11 protein family and its structure provides the first insight into the structural organization of this family. A number of fully and highly conserved residues defined by multiple sequence alignment of members of the Pfam01937.11 family were mapped onto the structure of At2g17340. The fully conserved residues are involved in the coordination of a metal ion and in the stabilization of loops surrounding the metal site. Several additional highly conserved residues also map into the vicinity of the metal-binding site, while others are clearly involved in stabilizing the hydrophobic core of the protein. The structure of At2g17340 represents a new fold in protein conformational space.

Structure at 1.6 A resolution of the protein from gene locus At3g22680 from Arabidopsis thaliana.

The gene product of At3g22680 from Arabidopsis thaliana codes for a protein of unknown function. The crystal structure of the At3g22680 gene product was determined by multiple-wavelength anomalous diffraction and refined to an R factor of 16.0% (Rfree = 18.4%) at 1.60 A resolution. The refined structure shows one monomer in the asymmetric unit, with one molecule of the non-denaturing detergent CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate} tightly bound. Protein At3g22680 shows no structural homology to any other known proteins and represents a new fold in protein conformation space.

The structure at 2.5 A resolution of human basophilic leukemia-expressed protein BLES03.

The crystal structure of the human basophilic leukemia-expressed protein (BLES03, p5326, Hs.433573) was determined by single-wavelength anomalous diffraction and refined to an R factor of 18.8% (Rfree = 24.5%) at 2.5 A resolution. BLES03 shows no detectable sequence similarity to any functionally characterized proteins using state-of-the-art sequence-comparison tools. The structure of BLES03 adopts a fold similar to that of eukaryotic transcription initiation factor 4E (eIF4E), a protein involved in the recognition of the cap structure of eukaryotic mRNA. In addition to fold similarity, the electrostatic surface potentials of BLES03 and eIF4E show a clear conservation of basic and acidic patches. In the crystal lattice, the acidic amino-terminal helices of BLES03 monomers are bound within the basic cavity of symmetry-related monomers in a manner analogous to the binding of mRNA by eIF4E. Interestingly, the gene locus encoding BLES03 is located between genes encoding the proteins DRAP1 and FOSL1, both of which are involved in transcription initiation. It is hypothesized that BLES03 itself may be involved in a biochemical process that requires recognition of nucleic acids.

The structure at 2.4 A resolution of the protein from gene locus At3g21360, a putative Fe(II)/2-oxoglutarate-dependent enzyme from Arabidopsis thaliana.

The crystal structure of the gene product of At3g21360 from Arabidopsis thaliana was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 19.3% (Rfree = 24.1%) at 2.4 A resolution. The crystal structure includes two monomers in the asymmetric unit that differ in the conformation of a flexible domain that spans residues 178-230. The crystal structure confirmed that At3g21360 encodes a protein belonging to the clavaminate synthase-like superfamily of iron(II) and 2-oxoglutarate-dependent enzymes. The metal-binding site was defined and is similar to the iron(II) binding sites found in other members of the superfamily.

Reduced anterior tibial translation associated with adaptive changes in the anterior cruciate ligament-deficient joint: goat model.

Adaptive changes in the menisci and adjacent posterior capsule were documented within anterior cruciate ligament-deficient knee (stifle) joints in the goat model. These physical changes in the menisci and capsule developed over time and were associated with reduction in the initial (time zero) abnormal anterior tibial translation following transection of the anterior cruciate ligament. At 50 N of applied force, the normal goat knee joint has a total anterior-posterior translation of 0.6+/-0.1 mm (+/- SEM) at 45 degrees of flexion and 0.3+/-0.1 mm at 90 degrees. The translation immediately after transection (time zero) with 50 N of force was 8.2+/-0.5 mm at 45 degrees and 4.9+/-0.9 mm at 90 degrees. Within 8 months after transection and at 50 N of force, the treated knees had reduced translation values of 5.3+/-0.6 mm at 45 degrees of flexion and 2.9+/-0.5 mm at 90 degrees, or 35 (p<0.001) and 40% reductions, respectively, compared with the values at time zero. Magnetic resonance images of the ligament-deficient stifle joints, as well as gross measurements and image analysis after dissection, consistently demonstrated increases in cross-sectional area and volume of the menisci compared with the contralateral controls. These secondary changes were most pronounced in the posterior portion of the medial menisci, and histologic evaluation demonstrated hypercellularity with the accumulation of poorly organized collagen, reduced safranin O staining (proteoglycan matrix synthesis), a thickened capsule and capsule attachment, and increased vascularity at the meniscal capsule interface.

Assessment of donor cell and matrix survival in fresh articular cartilage allografts in a goat model.

The long-term survival of allografts of articular cartilage has been proposed to be dependent on the survival of the cells that maintain the unique structural and material properties of the allograft. In this study, we assessed cell survival in 24 fresh articular cartilage allografts of the medial plateau in a Spanish-goat model. A DNA-probe technique was used to distinguish clearly between DNA from donor (allograft) and host cells. The intraarticular survival of viable allograft chondrocytes in the transplanted articular cartilage started to diminish as early as 3 weeks after transplantation; however, there was considerable variation in the amount of donor cell DNA detected in the allografts at 6 and 12 months following transplantation. This contrasts with our experience with fresh allografts of ligament, tendon, and meniscus, in which no donor DNA was detected 4 weeks after transplantation. DNA from host cells was present in all articular cartilage allografts, as evidenced by detectable unique host DNA patterns. Histological and histochemical assays showed that none of the transplants demonstrated normal structure and composition at 1 year after transplantation. The grafts in which large quantities of donor DNA were present appeared grossly superior to those with no or reduced remaining demonstrable donor DNA.

Characterization of the repair tissue after removal of the central one-third of the patellar ligament. An experimental study in a goat model.

UNLABELLED: The purpose of this study was to characterize the repair tissue that develops after removal of a portion of the patellar ligament for use as a graft. A six-millimeter-wide strip was obtained from the central portion of the patellar ligament with tibial and patellar bone plugs from one knee (stifle joint) of eight goats. The repair tissue that formed in the defect was characterized in terms of its structural, material, histological, and ultrastructural properties twenty-one months after the operation. The contralateral patellar ligament served as a control. Representative specimens were taken from the proximal, middle, and distal portions of the repair tissue and the control tissue for histological study and examination with transmission electron microscopy. The six-millimeter-long defect filled with repair tissue that increased the cross-sectional area by a mean of 42 per cent compared with the control values (p < 0.05). The maximum force to failure and the ultimate stress of the repair tissue were significantly decreased (by a mean of 51 and 65 per cent, respectively) compared with those of the controls (p < 0.001 for both). The stiffness also was reduced, by a mean of 27 per cent, but this was not significant (p > 0.05). Magnetic resonance imaging of the donor site showed slightly increased signal intensity compared with the intensity on the control side. Histological sections from the donor site contained collagenous (scar) tissue that was less organized, more cellular, and more vascular than the control tissue. Evaluation of the ultrastructure revealed that the repair tissue was composed primarily of collagen fibrils with a small diameter (range, fifty to 100 nanometers). CLINICAL RELEVANCE: The results of the present study suggest that the repair tissue that develops after removal of a strip of the patellar ligament for use as a graft is not comparable with normal tissue in terms of its structural, material, histological, and ultrastructural properties by twenty-one months. This should be kept in mind when this repair tissue is considered for use as a graft for revision of a reconstruction of the anterior cruciate ligament.

Survival of cells after intra-articular transplantation of fresh allografts of the patellar and anterior cruciate ligaments. DNA-probe analysis in a goat model.

The fate of donor cells in fresh allografts of the patellar and anterior cruciate ligaments was assessed after transplantation of the allografts as substitutes for the anterior cruciate ligament in goats. DNA-probe analysis was used to distinguish between the DNA of individual goats. Donor DNA was completely replaced by recipient DNA in both the transplanted patellar and anterior cruciate ligaments within a four-week period. Simultaneous full-thickness skin transplants in the same animals were not rejected during the interval of rapid loss of donor DNA from the allografts. The absence of rejection of the skin grafts at the one-week interval suggests that no pre-existing antibody associated with an immune reaction was responsible for the rapid loss of DNA in the allografts.

Spontaneous repair of full-thickness defects of articular cartilage in a goat model. A preliminary study.

BACKGROUND: Full-thickness defects measuring 3 mm in diameter have been commonly used in studies of rabbits to evaluate new procedures designed to improve the quality of articular cartilage repair. These defects initially heal spontaneously. However, little information is available on the characteristics of repair of larger defects. The objective of the present study was to define the characteristics of repair of 6-mm full-thickness osteochondral defects in the adult Spanish goat. METHODS: Full-thickness osteochondral defects measuring 6 x 6 mm were created in the medial femoral condyle of the knee joint of adult female Spanish goats. The untreated defects were allowed to heal spontaneously. The knee joints were removed, and the defects were examined at ten time-intervals, ranging from time zero (immediately after creation of the defect) to one year postoperatively. The defects were examined grossly, microradiographically, histologically, and with magnetic resonance imaging and computed tomography. RESULTS: The 6-mm osteochondral defects did not heal. Moreover, heretofore undescribed progressive, deleterious changes occurred in the osseous walls of the defect and the articular cartilage surrounding the defect. These changes resulted in a progressive increase in the size of the defect, the formation of a large cavitary lesion, and the collapse of both the surrounding subchondral bone and the articular cartilage into the periphery of the defect. Resorption of the osseous walls of the defect was first noted by one week, and it was associated with extensive osteoclastic activity in the trabecular bone of the walls of the defect. Flattening and deformation of the articular cartilage at the edges of the defect was also observed at this time. By twelve weeks, bone resorption had transformed the surgically created defect into a larger cavitary lesion, and the articular cartilage and subchondral bone surrounding the defect had collapsed into the periphery of the defect. By twenty-six weeks, bone resorption had ceased and the osseous walls of the lesion had become sclerotic. The cavitary lesion did not become filled in with fibrocartilage. Instead, a cystic lesion was found in the center of most of the cavitary lesions. Only a thin layer of fibrocartilage was present on the sclerotic osseous walls of the defect. Specimens examined at one year postoperatively showed similar characteristics. CONCLUSIONS: Full-thickness osteochondral defects, measuring 6 mm in both diameter and depth, that are created in the medial femoral condyle of the knee joint of adult Spanish goats do not heal spontaneously. Instead, they undergo progressive changes resulting in resorption of the osseous walls of the defect, the formation of a large cavitary lesion, and the collapse of the surrounding articular cartilage and subchondral bone. CLINICAL RELEVANCE: As surgeons apply new reparative procedures to larger areas of full-thickness articular cartilage loss, we believe that it is important to consider the potential deleterious effects of a "zone of influence" secondary to the creation of a large defect in the subchondral bone. When biologic and synthetic matrices with or without cells or bioactive factors are placed into surgically created osseous defects, the osseous walls serve as shoulders to protect and stabilize the preliminary repair process. It is important to protect the repair process until biologic incorporation occurs and the chondrogenic switch turns the cells on to synthesize an articular-cartilage-like matrix. It takes a varying period of time to fill a large, surgically created bone defect underlying a chondral surface. The repair of such a defect requires bone synthesis and the reestablishment of a subchondral plate with a tidemark transition to the new overlying articular surface. The prevention of secondary changes in the surrounding bone and articular cartilage and the durability of the new reparative tissue making up the articulating surface are issues that must be addressed in future studies.

The effects of in situ freezing on the anterior cruciate ligament. An experimental study in goats.

We developed an in situ freeze-thaw model designed to simulate an ideally placed and oriented autogenous graft of the anterior cruciate ligament. In this model, the anterior cruciate ligament was exposed, and the femoral insertion, tibial insertion, and body of the anterior cruciate ligament were frozen in situ with specially designed freezing probes. Freeze-thaw cycles were repeated five times. We used the technique in thirty-three mature goats to study the biological and biomechanical outcomes of the devitalized and devascularized anterior cruciate ligament at zero, six, and twenty-six weeks after treatment. Thus, the collagen fibers of the simulated autogenous graft remain in normal anatomical position and the simulated graft is fixed under physiological tension. At twenty-six weeks, no statistically significant differences were noted between treated and contralateral control (untreated) ligaments relative to anterior-posterior translation, maximum force to rupture, stiffness in the linear region of the force-length curve, modulus of elasticity in the linear region, strain to maximum stress, or maximum stress. The only statistically significant difference was an increase in cross-sectional area of the ligament. This increase was 22 and 42 per cent greater than that in the control ligaments at six weeks and six months. At six months, the ligaments in the control group had an average mid-cross-sectional area of 17.7 +/- 1.2 square millimeters and the ligaments in the experimental group, 25.2 +/- 3.1 square millimeters. Changes in the size and density of the collagen fibrils also were demonstrated at six months. These observations are in sharp contrast to our previous studies of replacement of the anterior cruciate ligament, in which an allograft of the ligament or an allograft supplemented with a 3M ligament augmentation device (LAD; 3M, St. Paul, Minnesota) was used. In those studies, an average reduction in maximum strength of 75 per cent for the allografts and 50 per cent for the allografts that had a ligament-augmentation device was found at one year. We concluded that devitalized, devascularized anterior cruciate ligaments do not lose strength if the anatomical position and the orientation of the collagen fibers are not altered.

Cell survival after transplantation of fresh meniscal allografts. DNA probe analysis in a goat model.

UNLABELLED: Fibrochondrocytes synthesize and maintain the extracellular matrix responsible for the distinctive material and structural properties of a normal meniscus. Viable meniscal cells are believed to be necessary for the long-term maintenance of these properties in meniscal allografts. The purpose of this study was to determine if the donor cells (fibrochondrocytes) survive after a fresh meniscal allograft transplantation. A DNA probe technique was used to clearly distinguish the DNA patterns in donor cells from the host cells in the Spanish goat. No remaining donor DNA could be demonstrated at 4 weeks in transplanted meniscal tissue; it was all of host origin. The host DNA content at 4 weeks approached or exceeded the amount present in the contralateral control meniscus. CLINICAL SIGNIFICANCE: The results of this study demonstrate that viable cells in medial meniscal allografts transplanted from one animal to another do not survive. Host cells rapidly repopulate the transplanted meniscus. There is no evidence these new cells will maintain on a long-term basis the extracellular matrix of the meniscus. The evidence in this paper, that the fibrochondrocytes do not survive transplantation, suggests further justification is necessary for using grafts with living cells. Allografts with living cells have an increased expense, more complicated surgical logistics, and have a higher potential risk of disease transmission.

Intraarticular reaction associated with the use of freeze-dried, ethylene oxide-sterilized bone-patella tendon-bone allografts in the reconstruction of the anterior cruciate ligament.

One hundred nine patients over a 3 year period underwent reconstruction for chronic ACL ruptures using a freeze-dried, ethylene oxide-sterilized bone-patella tendon-bone allograft. Seven patients (6.4%) developed a characteristic persistent intraarticular reaction. This reaction was characterized by persistent synovial effusion with collagenous particulates and cellular inflammatory response. Synovial biopsies in all cases showed a similar chronic inflammatory process, characterized by fibrin, collagen, and phagocytic cells. The intraarticular white cells were predominantly lymphocytes. Removal of the allograft resulted in resolution of the reaction in all of the patients. Three of the seven patients showed HLA conversion. Gas chromatography demonstrated detectable levels of ethylene chlorohydrin, a toxic reaction product of ethylene oxide, within the allograft and synovium 14 months following implantation of one graft. These seven cases presented strongly suggest a nonspecific or immune mediated response that must be further delineated. The use of freeze-dried, ethylene oxide-sterilized allografts using standard techniques cannot be recommended for reconstruction of the ACL.

Biologic remodeling after anterior cruciate ligament reconstruction using a collagen matrix derived from demineralized bone. An experimental study in the goat model.

A matrix of demineralized cortical bone was used to reconstruct the anterior cruciate ligament in the goat model. This graft underwent considerable site-specific remodeling and transformation from a Haversian system at time zero into a ligament-like structure at 1 year. This transformation included new bone formation filling the osseous tunnels and replacing the demineralized matrix, development of a ligament-like transition zone within the graft, and ligamentous collagen orientation with crimp in the intraarticular portion of the graft. One year after surgery, the mean anterior-posterior translation in the reconstructed stifle joints at 30 N of tibial loading was 2.1 +/- 0.4 (+/- SEM). The mean ultimate force to failure for the reconstructed ligament at 1 year was 474 +/- 146 N compared with the time-zero (initial) strength of the matrix of 73 +/- 9 N. The cellular repopulation of the graft had no associated inflammatory cells. The potential clinical significance of these findings includes 1) replacement of a collagen matrix with bone within the osseous tunnels, 2) establishment of a more physiologic fibrocartilage transition at the graft insertion site, 3) the time-zero structural properties of a collagen matrix increasing to more desired values with biologic remodeling, and 4) a sterile biologic allograft with essentially no long-term inflammatory response.

Meniscal repair supplemented with exogenous fibrin clot and autogenous cultured marrow cells in the goat model.

This study was undertaken to evaluate the placement of fibrin clot and cultured autologous marrow cells in surgically created, full-thickness, meniscal lesions in the avascular zone in 32 female Spanish goats. The menisci were repaired with two vertically oriented sutures (N = 8), exogenous fibrin clot was placed into the meniscal defect before placement of the two sutures (N = 8), fibrin clot plus cultured adherent bone marrow cells were placed in the defect (N = 8), or the meniscal lesions were left unrepaired (N = 8). On gross and manual inspection, meniscal lesions showed some degree of healing in all animals except for the eight unrepaired lesions. All the experimental specimens had decreased tensile strength compared with the contralateral control medial menisci. Ultimate load to failure, energy absorbed to failure, and stiffness were less than 40% of the controls for all groups. Histologic sections demonstrated focal cellular areas consisting of giant cells and macrophages in the repair sites. Our observations failed to demonstrate a statistically significant enhancement of healing with the use of exogenous fibrin clot compared with vertically oriented sutures alone. The addition of cultured adherent autologous bone marrow-derived cells in conjunction with the fibrin clot did not enhance the meniscal healing.

Cruciate reconstruction using freeze dried anterior cruciate ligament allograft and a ligament augmentation device (LAD). An experimental study in a goat model.

One ACL in each of 11 mature goats was replaced with a freeze dried bone-ACL-bone allograft and a ligament augmentation device (LAD). The LAD was released from its tibial fixation at 3 months postoperation. Biomechanical, microvascular, and histological changes were evaluated 1 year following implantation. The reconstructed knees had a significantly greater total AP translation (3.1 +/- 0.5 mm) (mean and SEM) than the contralateral controls (1.0 +/- 0.1 mm). Differences in primary AP translation were responsible for 59% of the difference in total translation, with only a 0.6 mm difference in secondary translation. Neutral stiffness in the reconstructive knee was 22% of control, while stiffness at 30 N of anterior force was approximately 35% of controls. Ligament stiffness in the linear region for the ACL allograft/LAD was 53% of the control value of 691 N/mm. The maximum load of the allograft/LADs was 1,052 +/- 145 N, or 43% of the contralateral ACL control strength (2,448 +/- 144 N). Five of the six allografts failed at the femoral insertion. Energy (39%) to maximum load was less for allograft/LADs than controls but elongation to maximum load was the same as control. Histologic evaluation of the allograft/LADs revealed soft tissue cellular ingrowth into the LAD in the extraarticular portions. No bony growth into the LAD was observed. The collagen fibers of the graft appear to be arranged in a longitudinal orientation although some areas show chaotic collagen fibers. Microangiography revealed a periligamentous and endoligamentous vascular pattern reminiscent of a normal ACL and complete revascularization of the bone plugs.