Synthetic nano-hydroxyapatite (nHAP) has drawn much attention of clinicians and researchers due to its excellent biocompatibility, osteogenic potential, osteoconductive nature and optimum biodegradability [1], [2], [3]. Hydroxyapatite (Ca10(PO4)6(OH)2) is a hexagonally structured primary component of bone and teeth that provides hardness and strength to the bony tissues [4]. Therefore, nHAP has been extensively used as a bone substitute and/or as an implant for different types of bone defects. Moreover, nHAP also bears affinity towards numerous pharmacological substances and thereby, allows passage of these molecules across to the target site, serving as a drug delivery agent. In addition, nHAP has also been utilized as coatings on prostheses, for chromatographic purification of recombinant human proteins, in water purification systems for removal of heavy metals, and as a photocatalyst in chemical reactions [5], [6], [7], [8], [9], [10]. Due to such a diverse range of applications, nHAP holds importance not only as a biomaterial for tissue engineering and regenerative medicine but it is also pivotal for other sectors of research and development.

Previous studies have extensively described various methods for the synthesis of nHAP, which bears close resemblance to natural nHAP [11], [12], [13]. Some of the commonly used methods are – combustion method, hydrothermal method, mechanochemical synthesis method, sol-gel technique (aging 24 hours), wet precipitation method (aging 24 hours), sonochemical/Hydrothermal (150 ◦C for 25 h in an electric oven), microwave method (400 W, 45 min), and microemulsion (aged for 24 h at room temperature) [14], [15], [16], [17], [18]. Out of all the aforementioned methods chemical precipitation is the most widely used technique for the synthesis of nHAP. In this method, i.e., wet chemical precipitation technique, nHAP nucleation takes for a few hours, followed by an aging step that involves the maturation of nHAP for at least 24 hours [19]. Hence, this method, despite its widespread popularity is a time-consuming process.

Lasers as tools have been explored in different research areas and demand for laser-based applications is continuously evolving with advancements in laser physics [20]. The laser as a synthetic technique provides an alternative solution that has the advantages of being rapid, scalable, environmentally friendly, cost-effective, and allowing in situ processing. Compared to traditional synthesis, laser-assisted processing allow for the direct synthesis of nanomaterials in both gas and liquid environments with less environmental impact and energy loss; particularly, the suitability for processing thermally sensitive substrates is more advantageous. In contrast, traditional wet chemical procedures, particularly as a synthetic process, can produce nanomaterials with distinct morphologies, but toxic or ecologically unfavourable reagents are usually used. In contrast, laser synthesis methods in a solution environment frequently use the target as a precursor, avoiding the use of toxic reagents, and nanomaterials with smaller particle sizes can be obtained by regulating the laser power, laser wavelength, laser focal length, laser pulse width, and laser frequency [21], [22]. Thermal treatment, also known as annealing, is a common approach for nanomaterial synthesis that is carried out in a furnace at a high temperature based on the thermodynamics of material production. However, this process has some drawbacks, including time consumption, high thermal power, and energy loss with sample dimensions that are significantly smaller than the heated volume. Till date, to the best of our knowledge, none of the reports have used nanosecond laser in the wet precipitation method for the synthesis of nHAP in the chemical reaction, and this study is the first to report utilisation of nanosecond laser for the synthesis of nanosized HAP. Majorly, nanosecond laser has been used for the nanoparticle fabrication from already synthesized hydroxyapatite and for coatings on biometals [23], [24], however, none of the studies have focused on using laser pulses as heat source in the chemical reaction of hydroxyapatite nucleation.

Bone defects can occur due to various reasons such as trauma, accidents, sports injuries, osteoporosis, and tuberculosis [25]. To fill these bone defects autografts are considered as the gold standard, however, donor site morbidity and limited donors restrict their use and demands for alternatives. Synthetic bone grafts based on polymers, ceramics, and their composites are in clinical use [26]. These bone grafts are mostly osteoconductive that allows the cell attachment and proliferation, however, osteoinductive materials would be a choice to help in bone healing process. For instance, bioactive molecules such as bone morphogenic protein (BMP) and zoledronic acid (ZA) have been used extensively in clinical settings [27], [28]. A plethora of studies and clinical reports have documented their use in bone graft substitutes for enhancing biological performance of bone fillers [29]. In the previous study from our lab, we have performed detailed studies regarding loading bioactive molecules in nHAP based composites for their enhanced biological performances to be used in the in vivo bone regeneration models [30], [31], [32], [33], [34]. Nano-hydroxyapatite has been shown to be a potential carrier for bone drugs such as BMP and ZA.

In the present work composite cryogel containing laser-assisted nHAP was synthesized. For nHAP synthesis, a non-contacting nanosecond pulsed laser was used in the chemical reaction of nHAP as a heat source. This heat is localized to a confined volume where the laser is focused resulting in instant hydroxyapatite nanoparticle formation. In comparison to conventional techniques, the involvement of laser has significantly reduced the time of synthesis. Laser synthesized nHAP has properties similar to that of conventional and natural HAP. With this technological advancement, we have obtained uniform rod-shaped high purity HAP nanoparticles with adequate biocompatibility, osteoconductivity, and biodegradability. Further, the in vivo biosafety and bioefficacy of laser-based synthesized nHAP based composite was confirmed via ectopic bone regeneration. Additionally, synthesized nHAP based composites have been loaded with BMPs and ZA to study their carrier capacity and bone formation in animal muscle pouch model.