Demineralized Dentin Matrix (DDM) As a Carrier for Recombinant Human Bone Morphogenetic Proteins (rhBMP-2).

A bone graft and bone graft substitute should have at least one of the following properties: it should be (1) osteogenic, (2) osteoinductive and/or (3) osteoconductive. In addition, bone graft substitutes should be biocompatible and bioresorbable as well as easy to use and cost effective. Autologous cancellous bone is the clinical gold standard in bone grafting procedures1, 4 and it has osteogenic, osteoinductive, and osteoconductive properties. Because of disadvantages associated with harvesting autologous bone graft material, such as requiring an additional operation and possible donor site morbidity, there is a need for an alternative in terms of enhancing the bone healing for the treatment of large bony defects. One possible option is a newly developed biomaterial, the demineralized dentin matrix (DDM). It is based on autogenous tooth dentin and is produced through demineralization. It is osteoconductive and osteoinductive due to the fact that dentin contains extracellular Type I collagen and various growth factors. Based on the demineralization process the factors stay available to the host environment. In 1965, Urist already showed the formation of ectopic bone after implanting DDM into muscle pouches in rodents. DDM is used for example in dental surgery in the treatment of extraction socket preservation and guided bone regenerations. It functions as a scaffold to support bone regeneration, but can also be used as a carrier for rhBMP-2. When DDM serves as a carrier, it combines the properties of the grafting material with those of the delivered substances. This chapter will present the experimental and clinical studies of DDM for rhBMP-2 carrier as well as alternatives of bone graft substitute.

Autogenous demineralized dentin matrix from extracted tooth for the augmentation of alveolar bone defect: a prospective randomized clinical trial in comparison with anorganic bovine bone.

OBJECTIVES: The aim of this study was to prospectively evaluate the clinical efficacy and histological outcome of the autogenous tooth graft material (AutoBT) compared to that of anorganic bovine bone (Bio-Oss® , Geistlich, Switzerland) in post-extraction alveolar bone augmentation. PATIENTS AND METHODS: A total of 33 graft sites in 24 patients were included in this study. AutoBT was used in 21 sites of 15 patients and Bio-Oss® was used in 12 sites of 9 patients for alveolar bone augmentation 2-4 weeks after dental extraction. Vertical dimension of grafted bone was measured both at the time of graft placement and at implant fixture placement after 6 months. Trephine cores were harvested for the histomorphometric evaluations during implant placement when feasible. The primary stability of implant fixture was also measured. RESULTS: Infection of graft material or graft bed was not observed and graft sites healed without any notable complications in both groups. The vertical dimensions of alveolar bone increased by 5.38 ± 2.65 mm in AutoBT group and 6.56 ± 3.54 mm in Bio-Oss® group at 6 months post-extraction. Histomorphometrically, new bone formation of AutoBT-grafted site was 31.24 ± 13.87% while that of Bio-Oss® was 35.00 ± 19.33%. The implant stability quotient (ISQ) of implants placed in AutoBT-grafted sites measured 72.80 ± 10.81 while those placed in Bio-Oss® -grafted sites measured 70.0 ± 12.86. There were no statistically significant differences between measurements of the two groups. CONCLUSION: Autogenous demineralized dentin matrix from extracted tooth grafted to extraction sockets for the augmentation of vertical dimension was as effective as augmentation using anorganic bovine bone. Both groups showed favorable wound healing, similar amount of implant stability, and histologically confirmed new bone formation. Thus, the results of this study suggest that autogenous tooth graft material is a viable option for alveolar bone augmentation following dental extraction.

Demineralized dentin matrix scaffolds for alveolar bone engineering.

From the point of view of implant dentistry, this review discusses the development and clinical use of demineralized dentin matrix (DDM) scaffolds, produced from the patient's own extracted teeth, to repair alveolar bone defects. The structure and the organic and inorganic components of DDM are presented to emphasize the similarities with autogenous bone. Studies of DDM properties, such as osteoinductive and osteoconductive functions as well as efficacy and safety, which are mandatory for its use as a bone graft substitute, are also presented. The clinical applications of powder, block, and moldable DDM are discussed, along with future developments that can support growth factor and stem cell delivery.

Guided Bone Regeneration Using Demineralized Dentin Matrix: Long-Term Follow-Up.

PURPOSE: This case report reviews the long-term clinical outcomes of using demineralized dentin matrix (autogenous tooth bone graft material [AutoBT]) in 5 cases that were first reported in 2010. MATERIALS AND METHODS: Cone-beam computerized tomography was used to measure the height and width of the graft to determine the change in bone area from immediately after surgery to final follow-up (average, 5 yr 5.8 months). Corticocancellous bone formation and marginal bone resorption also were evaluated histologically 3 to 6 months after grafting, which focused mainly on remodeling capacities. RESULTS: Decreases in buccal height and alveolar ridge width ranged from -0.4 to -3.3 mm and from -0.4 to -4.2 mm, respectively. The change in bone area ranged from -8.1 to -36.2%. Corticocancellous bone had formed and was maintained successfully except for 1 mm of buccal marginal bone resorption in 1 case followed for 6 years 7 months. CONCLUSION: AutoBT, which was first reported for guided bone regeneration, showed that the corticocancellous bone that had formed had been maintained successfully with an implant after an average follow-up of 5 years. Although the number of samples was small, the results were consistent with those of other short-term follow-up studies on AutoBT.

Guided bone regeneration using autogenous tooth bone graft in implant therapy: case series.

Recently, techniques have been reported that involve the preparation of extracted teeth from patients used as particulated bone graft materials for bone graft purposes. For implant placement and bone graft, autogenous teeth bone graft materials were used in 15 patients, and clinically excellent results were obtained. In histological examination, favorable bony healing by osteoconduction was observed.

Demineralized Dentin Matrix Incorporated with rhBMP-2 Composite Graft for Treating Medication-Related Osteonecrosis of the Jaw.

Medication-Related Osteonecrosis of the Jaw (MRONJ) is characterized by bone exposure in the oral and maxillofacial region for more than eight weeks in patients treated with anti-resorptive agents, immunosuppressants, or anti-angiogenic agents, without prior radiation therapy or metastatic disease to the jaws. Conservative treatments can control infection in mild cases, but surgical intervention is necessary for patients with severe symptoms. A 78-year-old female with a history of bisphosphonate treatment for osteoporosis presented with persistent pain, swelling, and malodor following implant placement in the upper right maxilla. SPECT/CT imaging revealed a high-risk hot spot in the right maxillary region. BIS-guided surgery using the Qray pen-C was performed, selectively removing red fluorescent bone tissue. The defect was grafted with HuBT incorporated with rhBMP-2. Postoperative follow-ups at 4, 7, and 14 months showed successful bone healing, transforming into a corticocancellous complex, and implant placement without MRONJ recurrence. Allogeneic demineralized dentin matrix (DDM) incorporated with rhBMP-2 demonstrates effective bone healing and implant placement following BIS-guided MRONJ surgery. This case supports the use of DDM/rhBMP-2 for tissue regeneration in MRONJ treatment, enabling successful prosthetic restoration without recurrence.

Bone grafts using autogenous tooth blocks: a case series.

OBJECTIVE: A case study was conducted to examine the clinical results and histologic healing of bone grafts performed using an autogenous tooth block (AutoBT block), which was developed recently and proprietary. STUDY DESIGN: Guided bone regeneration, extraction socket graft, sinus bone graft, and ridge augmentation were performed using autogenous tooth block graft material in 12 patients from March 2009 to June 2010. The clinical outcomes of each case were examined, and tissue specimens were collected from 1 case 2.5 months after the bone graft for histopathological analysis. RESULTS: All of the cases had successful bone graft results. One patient developed wound dehiscence after surgery, although favorable secondary healing was achieved. One implant resulted in osseointegration failure. A histopathologic examination was performed after 2.5 months and showed excellent bone healing due to osteoconduction. The AutoBT block was incorporated into the upper soft tissue, aponeurosis, and lower recipient bone. CONCLUSION: There were no notable complications associated with the bone transplant materials. The AutoBT block is clinically useful for a variety of bone grafts.

Incorporating Moldable Demineralized Dentin Matrix into Treatment for a Jaw Cyst.

The enucleation procedure is a standard treatment for jaw cysts; however, it often results in post-operative bony defects. These defects can lead to serious complications such as the risk of pathologic fracture and delayed wound healing, especially in the case of large cysts where there may be soft tissue dehiscence. Even in the case of smaller cysts, most cystic defects remain visible on postoperative radiographs and can be mistaken for cyst recurrence during follow-up periods. To avoid such complications, the use of bone graft materials should be considered. While autogenous bone is the most ideal graft material as it can be regenerated into functional bone, it has limitations due to the inevitable harvesting surgery. Many tissue engineering studies have been conducted to develop substitutes for autogenous bone. One such material is moldable-demineralized dentin matrix (M-DDM), which can aid in regeneration in cases of cystic defects. This case report highlights a patient who demonstrated the efficacy of M-DDM in bone healing for filling the cystic defect.

Effect of Gamma Irradiation on the Osteoinductivity of Demineralized Dentin Matrix for Allografts: A Preliminary Study.

Demineralized dentin matrix (DDM) treated with gamma irradiation (GR) has shown promising results as an allograft without any adverse effects in in vivo and clinical studies. The purpose of this study was to evaluate the effects of 15 and 25 kGy GR on the osteoinductive properties of DDM at extra-skeletal sites. As a control group, non-irradiated DDM powder was implanted into the right subcutaneous tissues of the dorsal thigh muscles of 20 nude mice. DDM powder irradiated with 15 and 25 kGy was implanted into the left side. After two and four weeks, the bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry. After confirming osteoblast- and osteoclast-specific activities by alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) staining, a histological analysis was performed to measure the new bone formation and the number of osteoblasts and osteoclast-like cells on the surface of the DDMs. Histomorphometry was used to calculate the new bone formation area on the surface of the DDM particles (DDMs). The BMD in all the groups increased from two and four weeks without statistically significant differences. The osteoblasts were dominantly activated on DDM without GR, and DDM treated with 25 kGy compared to DDM treated with 15 kGy. Among the groups, new bone formation was identified in all the groups at each time point. In conclusion, GR at doses of 15 and 25 kGy does not affect the osteoinductive properties of DDM powder.

Impact of Autogenous Demineralized Dentin Matrix on Mandibular Second Molar after Third Molar Extraction: Retrospective Study.

The purpose of this retrospective study was to evaluate bone healing after autogenous demineralized dentin matrix (DDM) grafts, focusing on the distal root of the mandibular second molar after the extraction of the third. We included retrospective data from 20 patients who had undergone molar extractions (15 male, 41.9 ± 12.0 years) between January 2020 and September 2022 and had DDM grafts implanted on the extraction socket, immediately ("immediate graft") or 6 weeks ("delayed graft") after the first surgery without primary closure. Patients who underwent grafting on only one side were used as the control group (n = 4). Bone defects at the mandibular second molar were measured preoperatively and 4 months after the graft surgery using cone-beam computed tomography (CBCT). Improvement of bone defect (i.e., the change in the bony defect pre- vs. postoperatively) was compared between the control and graft groups using the Wilcoxon Signed Rank test, and the difference between immediate and delayed grafts was analyzed with the Mann-Whitney U test. Complications such as infections or graft failure did not occur. Although pre-operative defects were smaller in the control than in the graft group (2.98 ± 1.77 and 10.02 ± 3.22 mm, p = 0.001), post-operative defects were similar in both (2.12 ± 0.59 and 2.29 ± 1.67 mm, respectively). The improvement ratio was not statistically significant in the control group (22.68 ± 15.36%) but a difference was observed in the graft group (76.70 ± 15.36%, p = 0.001). The amount of improvement of bone defect was not affected by graft timing or patient sex. In conclusion, DDM can improve bone defect at the distal aspect of the mandibular second molar after third molar extraction.

Allogeneic Demineralized Dentin Matrix as rhBMP-2 Carrier: A Retrospective Clinical Study.

PURPOSE: To compare the clinical outcomes of autogenous and allogeneic demineralized dentin matrices loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2; auto- and allo-DDM/rhBMP-2) by measuring the buccal marginal bone resorption around dental implants. MATERIALS AND METHODS: This retrospective study included patients who underwent dental implant placement with auto-DDM/rhBMP-2 as the control group and allo-DDM/rhBMP-2 as the experimental group. The primary outcome was buccal marginal bone resorption on CBCT. The resorption was calculated during T0 (from surgery to prosthetic loading), T1 (during the first year after loading), and T2 (during the second year after loading). The secondary outcome was the histologic analysis of five specimens of each group, obtained during the prosthetic procedure. RESULTS: Among the 103 implants, 61 and 42 implants were placed with auto- and allo-DDM/rhBMP-2 matrices, respectively. The resorptions of all periods were similar between the groups (T0: 0.65 ± 0.71 and 0.67 ± 0.81 mm, T1: 0.55 ± 0.60 and 0.59 ± 0.81 mm, and T2: 0.29 ± 0.45 and 0.20 ± 0.30 mm with auto- and allo-DDM/rhBMP-2, respectively). The histologic and histomorphometric analysis revealed similar osteoinductive aspects and proportions of new bone between the groups. CONCLUSION: Allo-DDM/rhBMP-2 showed comparable outcomes in terms of buccal marginal bone resorption to auto-DDM/rhBMP-2 during the second year after loading.

Review of Allogeneic Dentin Graft for Maxillofacial Bone Defects.

Although autogenous demineralized dentin matrix (auto-DDM) has shown promising clinical and histological results, it has certain limitations beyond its osteoinductivity and osteoconductivity. Therefore, the application of dentin graft material from other individuals-allogeneic DDM (allo-DDM)-has been considered an alternative to auto-DDM. However, few studies have investigated the osteoinductivity and antigenicity of allo-DDM. Herein, we reviewed all human studies related to allogeneic dentin application for the management of maxillofacial bone defects. Clinical studies have shown the osteoinductivity of allo-DDM in extraskeletal and skeletal sites, regardless of occasional antigenicity. Impact statement Although autogenous demineralized dentin matrix (auto-DDM) has shown promising clinical and histological results, it has certain limitations beyond its proven osteoinductivity and osteoconductivity. Therefore, the application of dentin graft material from other individuals-allogeneic DDM (allo-DDM)-has been considered as an alternative to auto-DDM. However, few studies have investigated the osteoinductivity and antigenicity of allo-DDM. This is the first review of all human studies related to allogeneic dentin grafts for the management of maxillofacial bone defects. Clinical studies have shown the osteoinductivity of allo-DDM in extraskeletal and skeletal sites, regardless of occasional antigenicity.

Dentin-Derived-Barrier Membrane in Guided Bone Regeneration: A Case Report.

An autogenous, demineralized, dentin matrix is a well-known osteo-inductive bone substitute that is mostly composed of type I collagen and is widely used in implant dentistry. This single case report describes a successful outcome in guided bone regeneration and dental implantation with a novel human-derived collagen membrane. The authors fabricated a dentin-derived-barrier membrane from a block-type autogenous demineralized dentin matrix to overcome the mechanical instability of the collagen membrane. The dentin-derived-barrier acted as an osteo-inductive collagen membrane with mechanical and clot stabilities, and it replaced the osteo-genetic function of the periosteum. Further research involving large numbers of patients should be conducted to evaluate bone forming capacity in comparison with other collagen membranes.

Allogeneic Dentin Graft: A Review on Its Osteoinductivity and Antigenicity.

Studies on allogeneic demineralized dentin matrix (Allo-DDM) implantation in the 1960s and 1970s provided the most reliable preclinical evidence of bone formation and antigenicity in an extraosseous site. Recently, applications of Allo-DDM at skeletal sites were studied, and have provided reliable evidence of bone-forming capacity and negligible antigenicity. However, the osteoinductivity and antigenicity properties of Allo-DDM in extraskeletal sites have not yet been investigated due to the lack of follow-up studies after the initial research. The clinical applications of autogenous DDM (Auto-DDM) have been standardized in some countries. Long-term clinical studies have reported the development of several shapes of Auto-DDM, such as powders, blocks, moldable forms, and composites, with recombinant human bone morphogenetic protein-2. For the development of Allo-DDM as a reliable bone graft substitute next to Auto-DDM, we reviewed preclinical studies on the bone induction capacity of allogeneic dentin at extraskeletal as well as skeletal sites. Electronic databases were screened for this review in January 2020 and searched from 1960 to 2019. This review aims to provide a foundation on the preclinical studies of Allo-DDM, which could enable future researches on its osteogenic capability and antigenicity. In conclusion, Allo-DDM showed great potential for osteoinductivity in extraskeletal sites with low antigenicity, which neither adversely affected osteogenic capability nor provoked immunologic reactions. However, the risk of viral disease transmission should be researched before the clinical application of Allo-DDM.

Effects of gamma irradiation on the measurement of hepatitis B virus DNA in dentin harvested from chronically infected patients.

BACKGROUND: The manufacturing of the demineralized dentin matrix (DDM) has been proven to extensively reduce the presence of human hepatitis B viral DNA (HBV DNA). This study measured and compared HBV DNA in fresh dentin to that in gamma radiation (GR)-sterilized dentin extracted from HBV-infected patients. The application of GR as a means of terminal sterilization is hypothesized to inactivate or eliminate HBV within the dentin matrix. METHODS: Dentin from 18 HBV-infected patients was collected and divided into three fragments. The first fragment was unaltered and used as the control group; the remaining two fragments were sterilized with gamma radiation doses of 15 or 25 kGy. DNA was extracted and purified from each fresh (control), and the GR-sterilized (experimental) dentin specimen and HBV DNA copy numbers were evaluated on the basis of the real-time polymerase chain reaction. The copy numbers were used to assess GR efficacy as a means of terminal sterilization for HBV inactivation or elimination. RESULTS: HBV DNA was detected in 66.67% of the fresh dentin specimens. The differences in HBV DNA levels between the fresh dentin and the GR-sterilized dentin were confirmed by the Wilcoxon signed-rank test for the doses of 15 and 25 kGy with P value of 0.012 and 0.010, respectively. Among the twelve HBV-DNA-positive fresh dentin samples, HBV DNA persisted in eleven after GR sterilization, yet the copy number was reduced to <10 (except for a single sample within each experimental group). CONCLUSIONS: The results suggest that 15 and 25 kGy of GR significantly reduced the HBV DNA levels in the fresh dentin matrix. Expansion of the possible clinical applications of allogenic grafts with the irradiated DDM will require additional studies, including validation of viral load inactivation to prevent infectious transmission and examination of GR exposure effects on the osteoinductivity of the matrix.

Histological Review of Demineralized Dentin Matrix as a Carrier of rhBMP-2.

In 2007, recombinant human bone morphogenetic protein-2 (rhBMP-2) was approved for use in humans at a concentration of 1.5 mg/mL with absorbable collagen sponges as an alternative to autogenous bone grafts for alveolar ridge augmentation, defects associated with extraction sockets, and sinus augmentation. However, the use of supraphysiological doses and the insufficient retention of rhBMP-2, when delivered through collagen sponge, result in dose-dependent side effects related to off-label use. Demineralized dentin matrix (DDM), an osteoinducing bone substrate, has been used as an rhBMP-2 carrier since 1998. In addition, DDM has both microparticle and nanoparticle structures, which do not undergo remodeling, unlike bone. In vitro, DDM is a suitable carrier for BMP-2, with the continued release over 30 days at concentrations sufficient to stimulate osteogenic differentiation. In this review, we discuss the histological outcomes of DDM loaded with rhBMP-2 to highlight the biological functions of exogenous rhBMP-2 associated with the DDM carrier in clinical applications in implant dentistry. Impact Statement Demineralized dentin matrix (DDM) has been used as an recombinant human bone morphogenetic protein (rhBMP-2) carrier and osteo-inducing bone substrate to facilitate continued release and stimulate osteogenic differentiation. In this review, we discuss the histological outcomes of DDM loaded with rhBMP-2 in order to highlight the biological functions of exogenous rhBMP-2 associated with the DDM carrier in clinical applications in implant dentistry.

Clinical application of autogenous demineralized dentin matrix loaded with recombinant human bone morphogenetic-2 for socket preservation: A case series.

BACKGROUND: Demineralized dentin matrix (DDM) has potential application as a carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2) in bone regeneration. PURPOSE: To evaluate the efficacy of DDM loaded with rhBMP-2 for socket preservation. MATERIALS AND METHODS: DDM loaded with rhBMP-2 (DDM/rhBMP-2) was applied to 10 experimental sites and DDM alone to 6 control sites. The changes in height and width of the extraction socket after preservation were measured by cone beam computed tomography. Trephine cores were harvested for histomorphometric evaluation before placement of the implant. RESULTS: The reductions in height and width of the socket were more significant in the group treated with DDM than in the group treated with DDM/rhBMP-2. The amount of new bone formation was 34.39% with DDM/rhBMP-2 and 29.75% with DDM; the respective amounts of residual dentin were 8.35% and 16.15%. Although the differences were not statistically significant, the dimensional changes, amount of bone formation, and replacement of DDM in DDM/rhBMP-2 with bone were superior to those of DDM alone. CONCLUSIONS: Within the limitations of this study, we suggest that DDM may be a potential carrier for rhBMP-2 and that it may be possible to reduce the rhBMP-2 concentration to 0.2 mg/mL.

Postulated release profile of recombinant human bone morphogenetic protein-2 (rhBMP-2) from demineralized dentin matrix.

Demineralized dentin matrix (DDM) has been used as a recombinant human bone morphogenetic protein-2 (rhBMP-2) carrier in many clinical trials. To optimize the clinical safety and efficacy of rhBMP-2 with DDM, efforts have been made to improve the delivery of rhBMP-2 by 1) lowering the administered dose, 2) localizing the protein, and 3) prolonging its retention time at the action site as well as the bone forming capacity of the carrier itself. The release profile of rhBMP-2 that is associated with endogenous BMP in dentin has been postulated according to the type of incorporation, which is attributed to the loosened interfibrillar space and nanoporous dentinal tubule pores. Physically adsorbed and modified, physically entrapped rhBMP-2 is sequentially released from the DDM surface during the early stage of implantation. As DDM degradation progresses, the loosened interfibrillar space and enlarged dentinal tubules release the entrapped rhBMP-2. Finally, the endogenous BMP in dentin is released with osteoclastic dentin resorption. According to the postulated release profile, DDM can therefore be used in a controlled manner as a sequential delivery scaffold for rhBMP-2, thus sustaining the rhBMP-2 concentration for a prolonged period due to localization. In addition, we attempted to determine how to lower the rhBMP-2 concentration to 0.2 mg/mL, which is lower than the approved 1.5 mg/mL.

Long-term follow-up of autogenous tooth bone graft blocks with dental implants.

Demineralized dentin matrix block (ABTB: Autogenous Tooth Bone Graft Block) is 3-D scaffold with same components and geometry with alveolar bone. ABTB is well incorporated and remodelled into cortico-cancellous bone with dental implant. The shape and volume were maintained with little marginal bone loss after average 44 months of follow-up.

Evaluation of the Healing Potential of Demineralized Dentin Matrix Fixed with Recombinant Human Bone Morphogenetic Protein-2 in Bone Grafts.

We aimed to evaluate the efficacy of demineralized dentin matrix (DDM) fixed with recombinant human bone morphogenetic protein-2 (rhBMP-2) through an experimental and a clinical study. Unilateral upper second and third premolars of eight beagles were extracted. A mucoperiosteal flap was elevated around the extraction socket, and a bone defect was made using a surgical drill. Each DDM was fixed with rhBMP-2, and autogenous bone was grafted at the bone defect area with a collagenous membrane. The beagles were euthanized at two, four, eight, and 12 weeks after receiving the bone graft. Block specimens involving grafted bone and surrounding natural bone were extracted. A total of 23 patients who received bone grafts using human DDM fixed with rhBMP-2 (AutoBT BMP) with implant placements (36 implants; maxilla: 14, mandible: 22) were selected. The implant stability, marginal bone loss, and clinical outcome were evaluated. Three trephine cores were harvested fourmonths after bone grafting, and histologic examination was performed. In the histological evaluation performed four weeks after the bone graft, autogenous bone showed 52% new bone formation and DDM fixed with rhBMP-2 showed 33% new bone formation. Twelve weeks after the bone graft, autogenous bone showed 75% new bone formation and DDM fixed with rhBMP-2 showed 48% new bone formation. In the clinical study, favorable osseointegration was obtained in 35 out of 36 implant sites (one case of osseointegration failure). In all cases, severe complications were not observed. Histomorphometrically, new bone formation was observed in 14.98% of the cases. The residual DDM particles were 6.22%. AutoBT BMP provides good osteoinductive and osteoconductive potential and clinical efficacy.