Table 2.

Studies on CB applications in animal-experiment-based bone regeneration and biomaterial assessment.

Study ID	Aim of the Study	The Methods				Finding/s	Limitations of the Study	Recommendation for Bone Regeneration
		Preparation of CB and Scaffold	Experimental or Commercial Materials	Methods of Characterisation	Preparation of the Animal
[41]	The aim was to create periosteum substitutes with exceptional osteogenic and angiogenic properties for treating rat calvaria bone defects	1. Preparation cube of the cuttlebone-derived organic matrix (CDOM) by removing calcium carbonate by using hydrochloric acid, phosphoric acid, and ethylenediaminetetraacetic acid and forming CDOM-HCl, CDOM-H3PO4, and CDOM-EDTA, respectively. 2. Forming “S”-oriented grooves on the surfaces.	Experimental material	1. SEM, EDS, FTIR. 2. Tensile test. 3. Water contact angle. 4. Biocompatibility test on mesenchymal stem cells. 5. Osteogenesis and Angiogenesis Assessment. 6. Micro-CT	1. Male rate of 300–350 g 2. An incision was made in the middle of the skull and stripped out the periosteum. 3. Bilateral defects of 5 mm were created and covered with different CDOM. 4. The suture was closed with the application of antibiotics.	1. CDOM films are highly biocompatible and the S groove is suitable for cell growth. 2. CDOM-EDTA exhibited the highest bone formation neovascularisation and bone regeneration and smaller amount of fibrous tissue (highest osteogenesis). 3. Biosafety of the CDOM films after surgery. 4. No inflammatory change or abnormality in the pathological section (histological safety of CDOM).	1. Mechanical limitations. 2. Eight-week interval.	A highly suitable candidate for clinical use in the regeneration of bone defects.
[42]	To test PVA-based biomimetic CB aerogel scaffolds (ASs) for osteogenesis in the calvarial bone defects of Sprague-Dawley (SD) IGS rats. (in vivo study)	1. The PVA solution one-step rapid freeze-drying method to obtain the PVA-CB AS. 2. PVA/MCNTs formed by adding (modified carbon nanotubes) MCNTs. 3. PVA/MCNT/HAp aerogel scaffolds were suspended in 50 mL of simulated body fluid (SBF) at 37 °C to promote the mineralization of HAp on the scaffolds.	Experimental material	1. TEM, FESEM, RS, TGA, and XRD. 2. Pore size estimation. 3. Compression test. 4. MC3T3-E1, ALP, and gene expression 5. Micro-CT analysis of the rats.	1. Eight-week-old male rats (5 rats in 4 groups). 2. The calvarial defects, 5 mm in diameter, were created on the calvarium using a trephine in each rat.	1. Pore size and MCNTs increased AS hydrophilicity and compression. 2. PVA/MCNT/HAp AS had the best cell adhesion, proliferation, ALP activity, target gene expression, and mineralisation. 3. Micro-CT showed bone development at eight weeks, with PVA/MCNT/HAp-filled bone defects.	Not mentioned.	PVA/MCNT/HAp AS has potential applications for bone regeneration.
[43]	To evaluate the cytotoxicity of the CB xenograft compared to commercial grafts (PerOssal®) (in vitro and in vivo study).	Hydrothermal reaction (HT) for preparing HAp from CB by mixing Aragonite and 0.6 M NH4H2PO4 to perform Ca/P; the mixture was heated at 200 °C for 12 h.	Experimental material	1. Biocompatibility (osteoblasts tests) MTT assay for the viability of hMSC. 2. XRD and SEM. 3. Body weight and temperature before and after implantation.	1. Total of 27 rabbits divided into three groups (CB, PerOssal®, and control group) 2. Bone graft material was injected into a femoral muscle in two experimental groups (CB + NaCl 0.9%) and Perossal® + NaCl 0.9%), with NaCl solely in the control group.	1. The non-toxic effect on hMSCs for both CB xenograft PerOssal® and pyrogenicity rabbit test was the same for both groups. 2. The properties of HAp from CB are similar to those of human HAp.	1. Only the acute phase (72 h) was measured, not the chronic phase 2. No bioavailability test was used. 3. No figure for XRD was shown.	CB material has potential application as bone graft material.
[44]	To determine the effect of HAp from CB as bone filler on femur bone regeneration in white rats (in vivo study).	Hydrothermal treatment (HT) was used to prepare HAp from CB by adding 1 M CaCO3 and 0.6 M NH4H2PO4, then heating the mixture at 200 °C for 12 h, followed by sintering at 900 °C for 1 h.	Experimental material	1. XRD. 2. Light microscope.	1. Thirty rats aged 3–4 months and weighing 200–300 g. 2. CB-HAp and bovine HAp powder (0.5 mg each) were inserted into the fractured femur bone defect (1 mm). 3. Treatment times were 28 and 56 days. 4. Three groups (control, bovine, and CB group).	1. The bone growth and regeneration process were affected by HAp-CB and the recuperation time (56 days). 2. HAp -CB and HAp of bovine bone provided the same result compared to the control group, so they can be used as bone fillers for fracture sites.	Not mentioned.	It can be used for regenerating bone.
[45]	To assess the potential of CB-derived HAp and any potential synergistic effects of platelet-rich plasma with this scaffold on bone healing. (In vivo study.)	CB block was treated with 5% NaClO for 48 h and 0.6 M of NH4H2PO4 to obtain Ca/P 1.67 and the mixture was heated at 200 °C for 24 h. Finally, CB was sterilised with irradiation.	Experimental material	1. XRD. 2. SEM. 3. Fluoroscopy and histopathological examination.	1. Platelet-rich plasma PRP was made from plasma and 8 mL of centrifuged blood. 2. Groups were randomly assigned to use both legs (tibia defect 3.5 mm): negative control, I with PRP, II with raw CB, III with raw CB and PRP, IV with CB/HAp, and V with CB/HAp and PRP. 3. Animals were killed eight weeks after implantation.	1. Raw CB is an acceptable bone defect filler by itself; however, processed HAp is more promising based on histopathologic terms of union and cortical indices. 2. CB is a reliable marine source that promotes the production of new bones.	A more extended evaluation period is needed to accurately evaluate this biomaterial and track the remodelling stage of the bone healing process.	It can be used for bone defect repair.
[46]	1 To evaluate the osteoconductivity of three HAp samples (natural, synthetic, and manufactured) using a sinus lift model in rabbits. (in vivo study). 2. To compare results to biomaterial topography and physical attributes.	1. Bovine hydroxyapatites [BHAp] and CB hydroxyapatite [CBHAp] with high calcined temperature) and synthetic hydroxyapatite (SHAp). 2. In this study, the particle sizes used were 250–1000 µm (BHAp and SHAp) and 500–1000 µm (CBHAp).	Commercial material	1. SEM. 2. BET for specific surface area analysis.	1. Twenty-four mature rabbits (3 kg) with 45-sinus floors. Insulin syringes were used to carefully introduce the substance into the sinus wall and membrane after administering antibiotics and anaesthetic. 2. The sample was separated into enlarged volume, SEM, and newly produced bone. 3. Each group included five samples, and results were assessed after 1, 5, and 12 weeks.	1. With natural HAp, more bone was detected than with synthetic HAp because they have bone-like macro-architectures with interconnected macropores. 2. With BHAp, there was a substantially increased bone-to-material contact, which improved the biomechanical environment.	More characters- action and investigation are needed to identify the biomaterial’s physicochemical properties.	Material–bone contact.
[47]	To demonstrate how the CB affects the speed of healing for long bone fractures.	Injection of the mixture of cuttlebone extract and NaCl 0.9% solution.	Experimental material	1. X-ray photo. 2. Callus index. 3. Histochemical examination.	1. Thirty-two rats were split into control and experimental groups. 2. The right tibia was broken after the incision. 3. Injections of CB and NaCl 0.9% were given to the treatment and control groups, respectively. 4. Leg was repositioned and secured.	CB sped up the healing of fractured bones by raising the synthesis of osteoblasts and forming thicker calluses.	1. Limited period for the study: 2–3 weeks. 2. No characterisation of CB HAp.	Improved bone healing.
[48]	To separate low-molecular-weight sulphated chitosan from CB and extract, purify, and test its anticoagulant (AC), cytotoxic, and antiviral (AV) activities on birds (in vitro study).	1. Researchers blended CB to powder after washing out mud and debris. Chitin was decalcified in a dilute of 1.82% HCl after NaOH and KMnO4, and oxalic acid deproteinized it. 2. Sulphated chitosan was obtained by mixing chitosan and chlorosulphonic acid. 3. The substance was dialysed, filtered, freeze-dried, and stored.	Experimental material	1. UV–vis spectroscopy. 2. Fluorescence spectroscopy. 3. FTIR, TGA, and FT-Raman spectra analysis. 4. Elemental analysis 5. DSC thermal analysis. 6. Measurement of the molecular weight. 7. NMR spectroscopy. 8. Anticoagulant assay. 9. Leucocyte migration inhibition assay. 10. Antiviral activity.	RBC button development was monitored during a 30 min incubation period at room temperature with 1% avian RBCs added to the mixture (SP-LMWSC diluted in PBS).	1. CB critical functional groups for SP-LMWSC were extracted. 2. The SP-LMWSC allows long AC sessions and inhibits avian LM. 3. SP-LMWSC prevents avian RBCs from hemagglutinating by binding to ND virus surface receptors. 4. SP-LMWSC’s in vitro AC, cytotoxic, and AV characteristics are promising for further investigations.	Not mentioned.	The material can be used for biomedical purposes and activities.
[49]	To generate a highly porous, nanoscale-sized HAp, a CB at high temperature was employed to improve protein adsorption and bone growth following subcutaneous implant. (In vivo study.)	1. CB blocks were immersed in 5% NaClO for two days to remove protein, then sealed in Teflon-lined st. st. reactors and 0.5 M (NH4)2HPO4 was added. 2. The high-temperature treatment was conducted at 180 °C for 96 h. Samples were collected at 12, 24, 48, and 96 h, then washed in boiling water and dried in an oven	Experimental material	1. SEM. 2. Mercury intrusion porosimetry. 3. XRD and FTIR. 4. Protein adsorption test. 5. MTS, ALP, and OCN tests. 6. Light microscope.	1. Twenty mice aged six weeks were included. Dorsal subcutaneous pockets were created and sterilised with 75% ethanol. A CB block was then implanted. 2. New bone growth was detected using light microscopy. 3. Each specimen had four portions analysed and four pictures selected for each section.	1. CB was utilised to make porous, nanocrystalline HA bone replacements in various shapes. 2. CB/HAp significantly enhances MSCs’ osteogenic phenotype without an osteogenic reagent. 3. High osteoinductive capacity affects protein adsorption and MSC differentiation.	Conducting a quantitative comparison of serum and CB/HAp concentrations is crucial.	It can be used as a bone replacement material.
[50]	To assess the biocompatibility of CB and CB-derived hydroxyapatite (CB/HAp). (In vivo study.)	1. CB1 was formed by defatting and freeze-drying and sterilisation 2. CB2 was obtained by removing organic components, washing, drying, and sterilizing. 3. CB/HAp hydroxyapatite was formed from CB2 using hydrothermal treatment at 200 °C 4. Coral-derived HAp was used to produce CHAp 5. CB block was 5 mm in diameter and 2 mm in thickness.	Experimental material	1. XRD. 2. Histologic investigations. 3. CCD camera-based digital image analysis system.	1. Twenty 9-week-old mice (22 ± 0.2 g) were used for aseptic surgical tech and anaesthesia for the back. 2. Through the incision, sterilised implants were placed subcutaneously with a probable dose of antibiotics.	1. Among the experimental implants, CB/HAp was the most biocompatible material with thinner fibrous capsule thickness. 2. CB2 preparation is more effective than CB1, but there is no difference between the two.	Short time interval: 2–4 weeks.	CB/HAp can be used inside the body.
[51]	To compare CB-implanted rabbit calvarial bone defect healing. (In vivo study.)	1. Defatting and freeze-drying (CB1). 2. Organic component removal (CB2). 3. High temperature at 200 °C/24 h for CBHAp (HAp from CB2). 4. Coral-derived HAp. 5. Hamster ovarian cell expression of CB1 together with rhBMP-2. 6. CB was formed into cylindrical disks with a diameter of 5 mm and a thickness of 2 mm	Experimental material	1. XRD. 2. Radiography. 3. Histological investigation.	1. Twenty-seven 9-month-old rabbits (3.2 ± 0.5 kg). 2. With an aseptic surgical procedure, a sagittal incision was made at the midline of the calvaria after anaesthesia and removal of the skin and periosteum was completed. 3. Antibiotic coverage was provided for three days	1. CBHAp may be more biocompatible for compact bone defect regeneration. 2. Osteoconduction makes CBHA a safe bone graft for compact bone defect models. 3. Group CBHAp had faster bone regeneration at 12 weeks.	Further experiments are needed to identify the characteristics of CB.	CBHAp is a valuable bone graft material with irregular bone defects.
[52]	To assess cuttlefish bone-derived HAp granules (material: CB-HAp) as alternative biomaterials for bone grafts (in vitro and in vivo study).	CB pieces were boiled with 4% NaClO to remove the organic components and HT was at 200 °C with (NH4)H2PO4 for Ca:P 10:6 and the Teflon pressure vessel. The size of CB HA granules ranged from 200 to 500 μm.	Experimental material	1. XRD and SEM. 2. Biological tests. 3. Micro-CT bone analysis.	1. Six adult rabbits (ages > three months) (weighing 2.5–3.0 kg) were anaesthetised and then three separates circular calvaria defects were made. 2. First defect: control; second defect: CB-HAp granules; third defect: pure HAp granules	1. Microporous CB-HAp granules, derived from raw CB, were produced through hydrothermal treatment. 2. Synthetic HAP is less biocompatible than CB-HAp. 3. Pure HAP does not regenerate bone like CB HAp granules. 4. None showed necrosis or foreign body responses.	More research is needed on the mechanical characteristics of CB-HAp granules.	CB-HAp may be used in bone graft substitutes for accelerated bone healing.
[53]	To evaluate the CB xenograft’s capacity for bone regeneration for 24 weeks using radiography and histology (in vivo study).	With the aid of a scalpel blade, the CB was reduced to tiny pieces and disinfected with ethylene oxide.	Commercial material	1. Physiological measurements. 2. Radiographical and biochemical evaluation. 3. Histological examination.	1. One hundred and five one-year-old rabbits were separated into five groups, then seven. 2. CB, demineralised bone matrix, bovine cancellous graft, and TCP filled a 3 mm uni-cortical defect after anaesthesia and periosteum excision (5 mm). 3. Broad-spectrum antibiotics were applied locally	1. Comparing CB to other groups, it was thought to be the best graft that was not rejected and caused no infection. 2. After one week, the fibrous union was initiated with the CB graft. 3. Twenty-eight days after surgery, the bone healing in the CB group progressed more quickly than in the other groups, indicating its effectiveness in orthopedic surgery.	Not mentioned.	CB appears to be a compatible material.
[54]	To synthesise microporous scaffolds by HT by converting CaCO3 in CB into calcium phosphate composites (HA and TCP) (in vitro and in vivo).	1. CB were cut into different dimensional cylinders with (NH4)2HPO4 were sealed in a Teflon SS autoclave in the furnace at 180 °C. 3. HT tested between 3 and 48 h in the pH of the solution was 7.8–8.2.	Experimental material	1. XRD, FTIR, and TGA. 2. Mechanical properties. 3. Hemotologial test (APTT), PT, TT. 4. Biological tests.	The prepared scaffold was implanted in the rabbit femur after being stained with eosin.	1. With tunnel-like microstructures, HT can perform CB scaffold for 3 h. 2. MTT showed that macroporous materials were non-toxic and proliferated. 3. Material biocompatibility was determined in vivo and in vitro.	The number of samples and the details of animal preparation were not mentioned	A material’s ability to replace bone was perfected.
[55]	To examine the effect of CB thermal burn (second-degree) injuries in rats and compare them with silver sulfadiazine (SSD) in an in vivo study.	CB was demineralised with HCl, then washed with distilled H2O, and precipitated with 4% NaOH for 24 h, followed by washing and drying. The ointment was mixed with white petroleum (Vaseline) with CB in a 6:4 ratio.	Commercial material	1. XRD and FTIR. 2. Microscope and blood test. 3. Lipid peroxidation assay 4. Cytokine measurement	1. Seven-week-old 200–250 g rats were used. After cleaning with ethanol, the exposed area on each rat was heated to 100 °C for 10 min to induce a second-degree burn. 2. The mice were randomly assigned to 12 groups: SSD (0.5 g), CB (0.5 g/cm2), white petroleum (0.5 g), and negative control. 3. The ointment was applied twice daily to each group.	1. CB is a potential new material to treat wounds like skin burns as it reduces the inflammation of lipid peroxidation In addition, it promotes re-epithelialisation. Moreover, it accelerates wound healing.	No contribution for XRD.	It can be used for skin injury.
[18]	To create porous HAp from CB that has undergone high temperature and assess its biocompatibility using undecalcified materials (in vivo study).	CB block sinking in 4% NaOCl to remove organic compounds; after washing, 2 M (NH4)2HPO4 was added for 16 h at 180 °C, then immersed again in 2 M (NH4)2HPO4 and treated at 200 °C for 24 h hydrothermally + dried at 90 °C.	Experimental material	XRD, SEM, and TEM.	Rabbits weighing 2.5 kg were prepared with a 5 mm hole diameter made in the femoral condyles, 5–7 mm-long specimens were implanted, and a TEM examined the stained section.	With a good calcium source structure in the CB scaffold, the porous HAp can be used as a biomaterial for bone substitutes as it is biocompatible.	The number of samples was not specified, and the study required additional characteristic tests.	Biocompatibility is to be used as a suitable bone substitute.

Abbreviations: TEM, transmission electron microscope; FESEM, field emission scanning electron microscopy; XRD, X-ray diffraction; TGA, thermal gravimetric analysis; SEM, scanning electron microscopy; FTIR, Fourier-transform infrared.